A co-stimulatory signal through ICAM-beta2 integrin-binding potentiates neutrophil phagocytosis.

The beta2 integrin LFA-1 (lymphocyte function associated antigen; CD11a/CD18) is the common ligand for the intercellular adhesion molecules (ICAMs). Integrins support cell function by providing co-stimulatory second signals that are a precondition for full cell activation first described for ICAM-1-binding to LFA-1 in lymphocytes. Integrins can also serve to activate functions associated with distinct subunits of other integrins. In addition to LFA-1, neutrophils express the beta2 integrin Mac-1 (CD11b/CD18; CR3) that apparently contains multiple sites that bind invading microbes directly or through surface-fixed C3, resulting in activation of the phagocyte function. Expression of the LFA-1 counter-receptor ICAM-1 on endothelial cells occurs only at the site of inflammation. Therefore, in neutrophils, ICAM-1 ligand binding could, as with lymphocytes, also play a part as a co-stimulatory signal to induce full phagocytotic function. We show that in neutrophils, the LFA-1 ligand interaction is the stimulatory signal to express full phagocytotic activation. This is best demonstrated by the rapid association of Streptococcus pyogenes with neutrophils, followed by ingestion, strong oxidative-burst induction and enhanced killing of these bacteria, which are well-known for their resistance to human neutrophil defense. These findings may contribute to the development of therapeutic strategies targeting the modulation of ICAM-1-leukocyte interaction.

Epidemiology of invasive Streptococcus pyogenes infections in Germany, 1996-2002: results from a voluntary laboratory surveillance system.

A nationwide voluntary laboratory-based surveillance study of invasive Streptococcus pyogenes (group A streptococcus; GAS) infections was conducted in Germany between 1996 and 2002. Demographical and clinical information concerning the patients was obtained from the medical files. Multiple logistic regression analysis was used to determine risk-factors for fatal outcome. Invasive isolates were obtained from 475 patients, with 251 (52.8%) of the isolates cultured from blood. The most frequent emm types were emm1 (36.4%), emm28 (8.8%) and emm3 (8%). The speA, speC and ssa genes were present at variable frequencies in different emm types. The highest frequencies of speA and speC were found in emm1 (speA, 93.6%) and emm4 (speC, 94.7%), respectively. The estimated annual incidence of invasive GAS disease for 1997-2002 was 0.1 cases/100 000 individuals. This apparently low incidence rate might be explained by the voluntary nature of the surveillance system, resulting in relatively few cases being referred to the laboratory. Complete clinical information was available for 165 cases. The overall case fatality rate was 40.6%, and was highest (65.2%) in the group aged 60-69 years. Shock, an age of >or=30 years and adult respiratory distress syndrome were predictors of a fatal outcome in a multiple logistic regression analysis. Overall, 6.7% of the cases were considered to be nosocomial, and nine cases of puerperal sepsis were observed. The study underscores the importance of invasive S. pyogenes disease in Germany.

Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS).

Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de.

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.

Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials.

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (>70% of positive control) accompanied by increased adhesiveness (>60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (<10%) accompanied by a low percentage of adhering neutrophils (<10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

Estimation of type-specific anti-streptococcal M-protein antibodies with biotinylated peptides in human sera.

The design of a novel enzyme-linked immunosorbent assay for the estimation of antibodies directed against the N-terminus of the M-protein of Streptococcus pyogenes is described. The ELISA employs biotinylated peptide antigens of the types 1, 4, 12 and 19 immobilized by (strept-)avidin on the surface of polystyrene microtiter wells. In rabbit hyperimmune sera and in human serum samples, antibodies against the corresponding serotype could be detected with high sensitivity and specificity.

Implementing and evaluating a rotating surveillance system and infection control guidelines in 4 intensive care units.

BACKGROUND: In clinical practice, scientific evidence about infection control is often ignored and hygiene rituals are followed. METHODS: Within an evidence-based infection control program, a quarterly rotating surveillance program for nosocomial infections was implemented in 4 intensive care units (ICUs) at the Aachen University Hospital, Germany. RESULTS: For the first time, the unit-specific nosocomial infection situation was made clear to the clinical staff by interpretive feedback of the surveillance data. This led to an increased awareness of infection control and a critical review of hygiene practices. After the first surveillance period, the hygiene practices of each ICU were revised and modified. The Centers for Disease Control and Prevention/Hospital Infection Control Practices Advisory Committee guidelines for the prevention of nosocomial infections were adopted and established in tight collaboration with the ward staff. CONCLUSIONS: Within the surveillance process, communication and team spirit between infection control and patient care personnel showed a remarkable improvement. Awareness and compliance with hospital hygiene and infection control practices could be raised without directive interaction.

Macrolide resistance in Streptococcus pyogenes isolates from throat infections in the region of Aachen, Germany.

Macrolide-resistance was assessed in 216 consecutive Streptococcus pyogenes isolates from throat infections in the region of Aachen, Germany. Seventeen isolates were resistant to erythromycin: 12 isolates revealed a macrolide (M) phenotype and harbored mefA, and five strains expressed an inducible macrolide-lincosamide-streptogramin B (MLSB) phenotype of which four strains harbored ermA(TR) and one strain contained ermB(AM). Telithromycin (HMR 3647) and quinupristin/dalfopristin remained active particularly against the ermA(TR)-containing S. pyogenes isolates studied. Random amplified polymorphic DNA analysis identified multiple clones among erythromycin-resistant strains, but did not discriminate beyond the emm-type. mefA was present in three isolates either with emm2, emm12, or emm75, and in nine isolates with emm4. All four strains with ermA(TR) contained emm77, and the single strain with ermB(AM) harbored emm1. Despite the relative low rate of macrolide-resistance, these data suggest that at least three different macrolide-resistance determinants are prevalent in Germany and that mefA has spread rapidly into multiple clones of S. pyogenes.

Discriminative power of fatty acid methyl ester (FAME) analysis using the microbial identification system (MIS) for Candida (Torulopsis) glabrata and Saccharomyces cerevisiae.

Candida (Torulopsis) glabrata is frequently isolated in cases of fungal infection and commonly shows acquired or innate fluconazole resistance. Saccharomyces cerevisiae, an emerging opportunistic yeast pathogen, causes serious systemic infections in immunocompromised, and vaginitis and superficial infections in immunocompetent patients. For both species reliable identification in the routine laboratory is mandatory, but species identification of strains, e.g. trehalose-negative C. glabrata, may be difficult. Therefore, gas-liquid chromatography (GLC) of whole cell fatty acid methyl ester (FAME) profiles, that is independent of assimilation profiles of strains and suitable for reliable and rapid identification of clinically important yeasts, was applied. However, frequent misidentification of C. glabrata as S. cerevisiae has been reported when using the Yeast Clinical Database of MIS. Accuracy of MIS identification may be strongly influenced by the amounts of cell mass analyzed. Therefore, the present study compared the MIS results of these two yeasts achieved with different cell masses. Primarily we optimized, especially with respect to cost-effectiveness, the recommended streaking technique yielding a maximal recovery of 90-130 mg of cell mass from one plate, enabling testing of poor growing strains of C. glabrata. For all C. glabrata strains tested (n = 10) the highest identification scores (SI [Similarity Index] range 0.525-0.963, median 0.832) were achieved with 30 to 45 mg of cell mass. Only 5 of 10 S. cerevisiae strains revealed good library comparisons (SI > or = 0.5) when using 30 mg of cell mass, whereas with 45 mg all strains but two revealed this SI-level. For S. cerevisiae a higher amount of cell mass processed (up to 90 mg) was correlated with better identification scores (SI range using 90 mg: 0.464-0.870, median, 0.737). Several passages prior to FAME analysis of C. glabrata strains on recommended media revealed narrowing of SI ranges, but differences in SI values were not statistically significant.

Novel microtiter plate format for testing germ tube formation and proposal of a cost-effective scheme for yeast identification in a clinical laboratory.

The germ tube test is most widely used for presumptive identification of Candida albicans. Conventional testing is relatively time-consuming due to the hands-on time involved in preparing and viewing each isolate. In order to reduce work-load and costs we have developed a novel microtiter plate test format that offers several advantages: (i) use of removable strips of microtiter wells placed in lockwell frames, (ii) only one pipetting step for each isolate, (iii) direct micromorphological evaluation using an inverse microscope, (iv) use of a novel synthetic germ tube test medium, (v) reduction of the inoculum, permitting testing of minute colonies, (vi) thus, testing of different colonies in potentially mixed primary cultures of clinical specimens is encouraged and facilitated. Implementing this microtiter based germ tube test with simultaneous trehalase test for presumptive identification of Torulopsis (Candida) glabrata, we propose an identification scheme including this test format. This has been implemented in our routine laboratory permitting cost-effective presumptive identification of almost all clinically relevant yeast species.

The cyl genes of Streptococcus agalactiae are involved in the production of pigment.

The cyl genes of Streptococcus agalactiae are required for the production of hemolysin. Based on the observation that nonhemolytic S. agalactiae mutants do not produce pigment, a close genetic linkage between hemolysin and pigment has been postulated. To investigate this genetic linkage and to identify genes involved in the production of the S. agalactiae pigment, we screened mutant libraries for nonpigmented clones. Four distinct mutants were isolated with a nonpigmented and nonhemolytic phenotype. The mutations had occurred either in known cyl genes or in two open reading frames located immediately downstream. These novel genes are cotranscribed with the cyl gene cluster and were designated cylF and cylI. Our data indicate that identical genes participate in the production of S. agalactiae hemolysin and pigment.

Phagocytosis, oxidative burst, and killing of Candida dubliniensis and Candida albicans by human neutrophils.

Candida dubliniensis is a phylogenetically closely related species to Candida albicans. So far virtually nothing is known about the virulence factors of C. dubliniensis. Cell surface hydrophobicity (CSH) plays a critical role in adhesion of microorganisms to phagocytic cells; hydrophobic cells of C. albicans have been reported to be less sensitive to phagocytic killing than hydrophilic cells. C. dubliniensis displays CSH at 37 degrees C in contrast to C. albicans. To elucidate this issue, we determined levels of phagocytosis, oxidative burst and killing by human neutrophils of C. dubliniensis (n=10) compared to C. albicans (n=10) both cultured at 37 degrees C. Obtained test results revealed no statistically significant differences between these two yeast species for the level of phagocytosis (77.3 vs. 76.2% after 60 min), evoked oxidative burst (64.5 vs. 67.3% after 30 min) and killing (72.7 vs. 73.1% after 240 min). Therefore, human neutrophils can be considered to be equally efficient against these two yeast species.

Studies on streptococci resembling Streptococcus milleri and on an associated surface-protein antigen.

Ninety-nine strains of streptococci were isolated from 97 cases of pyogenic infections, most of which involved the teeth. Physiological and serological tests were performed on these streptococci and on 37 strains of streptococci from culture collections. The results were used for a numerical classification. Seventy-nine of the strains isolated from patients formed a cluster with Streptococcus milleri and group-F reference strains, and were therefore considered as streptococci resembling S. milleri. By the use of an antiserum prepared against strain Z3, protein antigens were demonstrated in acid extracts of 65% of the strains of S. milleri. These antigens were in only five strains not included in the S. milleri cluster.

High-level fluoroquinolone resistance in a clinical Streptoccoccus pyogenes isolate in Germany.

An isolate of Streptococcus pyogenes isolated from a 63-year-old woman with a serious wound infection was found to be highly resistant to fluoroquinolones (levofloxacin MIC > or = 32 mg/L). DNA amplification and sequencing revealed a serine-81 to phenylalanine substitution in gyrA and three substitutions in parC: serine-79 to phenylalanine, aspartic acid-91 to asparagine, and serine-140 to proline. To our knowledge, this is the first report from a European country of a clinical isolate of S. pyogenes with high-level fluoroquinolone resistance.

Distribution of multi-resistant Gram-negative versus Gram-positive bacteria in the hospital inanimate environment.

We prospectively studied the difference in detection rates of multi-resistant Gram-positive and multi-resistant Gram-negative bacteria in the inanimate environment of patients harbouring these organisms. Up to 20 different locations around 190 patients were surveyed. Fifty-four patients were infected or colonized with methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (VRE) and 136 with multi-resistant Gram-negative bacteria. The environmental detection rate for MRSA or VRE was 24.7% (174/705 samples) compared with 4.9% (89/1827 samples) for multi-resistant Gram-negative bacteria (P<0.001). Gram-positive bacteria were isolated more frequently than Gram-negatives from the hands of patients (P<0.001) and hospital personnel (P=0.1145). Environmental contamination did not differ between the intensive care units (ICUs) and the general wards (GWs), which is noteworthy because our ICUs are routinely disinfected twice a day, whereas GWs are cleaned just once a day with detergent. Current guidelines for the prevention of spread of multi-resistant bacteria in the hospital setting do not distinguish between Gram-positive and Gram-negative isolates. Our results suggest that the inanimate environment serves as a secondary source for MRSA and VRE, but less so for Gram-negative bacteria. Thus, strict contact isolation in a single room with complete barrier precautions is recommended for MRSA or VRE; however, for multi-resistant Gram-negative bacteria, contact isolation with barrier precautions for close contact but without a single room seems sufficient. This benefits not only the patients, but also the hospital by removing some of the strain placed on already over-stretched resources.

A new technique for quantitative bacterial assessment on burn wounds by modified dermabrasion.

Bacterial colonization and invasive bacterial infection is still one of the major problems in the treatment of burn victims. The standard procedures of bacterial monitoring of the burn would are i) swab-culture which is non-invasive but detects bacteria at the very surface and ii) biopsy-culture which gives a more complete view but has the disadvantage of being invasive. Therefore we developed a new technique for examination of microbial colonization of the wound surface. Dermabrasion of the upper layers of the wound was performed using a small rotating carbon-steel disc of defined roughness. The tissue samples obtained were analysed for bacterial growth in different culture media. Results were qualitatively and quantitatively compared with those of standard techniques performed in parallel. Our results show that this new technique is superior to the swab culture in identifying different bacterial species. The results can be compared with the biopsy technique, but has the advantage of being less invasive.

Assessment of acid production by various human oral micro-organisms when palatinose or leucrose is utilized.

One promising way of reducing caries is by using sucrose substitutes in food, e.g., palatinose or leucrose. Previous experiments addressing cariogenic potential of sucrose substitutes have focused mainly on Streptococcus mutans. However, given the many other micro-organisms in the oral cavity, this study compared the acid production of 100 bacterial strains representing 44 different species, by batch fermentation in a test tube containing, as a sole carbohydrate source, glucose, sucrose, palatinose, or leucrose. Selected strains were further analyzed in a fermenter. Additionally, 30 yeast strains were tested by an auxanographic sugar assimilation test. Only Lactobacillus spp., Stomatococcus mucilaginosus, Leuconostoc mesenteroides, and Weissella paramesenteroides, and some of the yeasts studied-i.e., Candida albicans, C. tropicalis, C. parapsilosis, and Saccharomyces cerevisiae-utilized leucrose and/or palatinose well. Strikingly, Stomatococcus mucilaginosus produced water-insoluble polysaccharides by fermentation of leucrose and palatinose. In the fermenter, the respective sucrose substitutes were not only cleaved but also utilized. Thus, extracellular cleavage by autochthonous micro-organisms may produce cariogenic cleavage products (glucose, fructose) that can be used by other well-characterized cariogenic bacteria found in the oral flora. Therefore, the anticariogenic potential of sucrose substitutes in food might be limited.

Inhibitor of complement, Compstatin, prevents polymer-mediated Mac-1 up-regulation of human neutrophils independent of biomaterial type tested.

The inflammatory reaction after cell contact with polymer materials is primarily mediated by activated neutrophils and may, in some cases, lead to exhaustion of neutrophil cell function. A direct consequence of this can be impairment of local or even systemic host defense mechanisms, which in turn can result in foreign body infections. Neutrophil activation, as indicated by the up-regulation of the Mac-1 adhesion receptor, is a reliable parameter for estimating the inflammatory risk due to implanted biomaterials. Because at blood contact, biomaterials immediately acquire a material-specific layer of blood proteins on their surface, including fibrinogen, complement, and immunoglobulin G, it is generally believed that after biomaterial contact, neutrophil activation primarily occurs by interaction with this protein layer. In this study, using our recently established polymer bead in vitro assay, we investigated whether complement inhibition alone can reduce biomaterial-mediated neutrophil activation, independent of the type of polymer and, hence, also its surface chemistry. Complement inhibition was achieved by using Compstatin, a recently developed complement inhibitor that binds to the complement component C3 preventing C3 convertase formation. We revealed significantly reduced (p < or = 0.025) Mac-1 receptor expression levels after 45 min of blood contact with the following polymers (without and with Compstatin): 1. polyurethane, 98.3%, 13.6%; 2. polymethylmetacrylate, 88.5%, 11.0%; and poly-D,L-lactide, 71.8%, 8.4%. Although these three polymer types acquire material-specific protein layers because of their different surface chemistry, complement inhibition by Compstatin alone proved to be sufficient to reduce neutrophil activation after surface contact, thus reducing the risk of biomaterial-mediated inflammatory reaction.

Increased titers of antibodies against streptococcal M12 and M19 proteins in patients with Tourette's syndrome.

It has been suggested that a post-streptococcal autoimmune process may be involved in the pathogenesis of a subgroup of children with tics and obsessive-compulsive symptoms (PANDAS). Elevated antibody titers against streptococcal antigens have also been described in adult patients suffering from Tourette's syndrome (TS). In order to characterise further streptococcal antigens, we focussed on M proteins. M proteins are a major virulence factor of group A streptococci and known to evoke an immunologic cross-reaction with diverse epitopes of human tissue including brain tissue. Therefore, antibodies against M proteins may play a role in the pathophysiology of at least a subgroup of TS patients. Antibodies against M proteins were studied in 25 adult patients suffering from TS and 25 healthy controls after careful medical examination. The antibody titers against the peptides M1, M4, M6, M12 and M19 were estimated by ELISA. Our results show increased titers of antibodies against the streptococcal M12 and M19 proteins in TS patients as compared with controls, while antibody titers against M1, M4 and M6 did not differ between the TS and control groups. Elevated serum titers of antibodies against M12 and M19 proteins support the view that a streptococcus-induced autoimmune process may be involved in TS. The finding of a possible autoimmune origin of TS has implications for both pathophysiology and future therapeutic strategies.

PCR reaction and dot-blot hybridization to monitor the distribution of oral pathogens within plaque samples of periodontally healthy individuals.

The purpose of this study was to determine the distribution of the putative periodontal pathogens Prevotella intermedia, Prevotella nigrescens, the three oral Capnocytophaga species (C. ochracea, C. sputigena, C. gingivalis), as well as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in plaque samples of periodontally healthy individuals. We chose a newly developed 16S rDNA directed polymerase chain reaction (PCR) and a previously described dot-blot hybridization assay to detect, differentiate, and quantify these bacteria directly in clinical samples. The subjects of these investigations were 66 sulcus fluid samples from 17 children (ages 3 to 5) attending a kindergarten, 48 sulcus fluid samples from 12 children (ages 9 and 10) from a primary school, and 25 subgingival plaque samples isolated from 6 different periodontally healthy dental students (ages 24 to 27). We were able to demonstrate the presence of P. nigrescens in 54 (kindergarten: 5; primary school: 33; students: 16) samples by PCR and quantified it by dot-blot hybridization. In addition, we found C. ochracea in 12 (kindergarten: 2; primary school: 10) samples by PCR reaction only. The other tested bacterial species were absent by the methods used. Furthermore we confirmed the specificity of our P. nigrescens-PCR in selected samples by enzyme electrophoresis.