An ability to interact with plasminogen or plasmin could provide micro‐organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen‐binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli . DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl‐terminal unrepeated region that were typical of M and M‐like proteins. Though the amino‐terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M‐like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen‐binding M‐like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M‐like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen‐activator and proteins exposed on the bacterial surface.
Background: In order to limit the use of broad-spectrum antibiotics, standardized empirical therapy against acute bacterial infections has been advocated. Methods: Guidelines for acute bacterial infections recommending increased usage of benzylpenicillin and restricted use of fluoroquinolones and cephalosporins have been implemented in Kalmar County, Sweden. We evaluated this strategy by recording therapy in patients with bacteraemia, antibiotic requisition, and point prevalence surveys prior to this intervention and at 6 and 12 months after. Results: Comparing the methods simultaneously, there was good agreement between them and an overall significant change in antibiotic usage. There was a significant shift from cefuroxime to cefotaxime and a borderline significant increase in the use of benzylpenicillin (p = 0.057). Based on the defined daily dose (DDD), a highly significant decrease in total cefotaxime and cefuroxime usage was observed that was not detected when applying the prescribed daily dose (PDD), which is adapted to local treatment practices. No change was found in mortality in Staphylococcus aureus bacteraemia or the incidence of Clostridium difficile infection. Conclusions: We conclude that the implementation of the new guidelines has resulted in a significant change in antibiotic usage, which could be conveniently monitored by antibiotic requisition if PDD is used in addition to DDD.
The aim of this study was to evaluate the usefulness of borrelia serology (Quick ELISA C6 Borrelia assay kit) as a diagnostic tool in cases of suspected neuroborreliosis. A retrospective patient material consisting of 124 paired serum and cerebrospinal fluid samples with a positive anti-borrelia antibody index (AI) using the IDEIA Lyme Neuroborreliosis test was compared with 124 AI-negative matched control subjects. The patients were divided into four groups based on presence of pleocytosis and age above or below 12 years. The presence of positive C6 serology in AI-positive patients with pleocytosis was 89% (83/93), significantly different (p<0.01) from in patients without pleocytosis (58%, 18/31). In AI-positive patients aged > or =12 years with pleocytosis, 94% (51/54) had a positive C6 serology. Of AI-positive patients with a symptom duration of more than 30 days, 93% (27/29) were positive by the C6 test. We conclude that the C6 serum test, together with clinical evaluation, is a powerful diagnostic tool in adult (> or =12 years) European patients with suspected neuroborreliosis with a symptom duration of more than 30 days. Patients with suspected neuroborreliosis and positive C6 results should be further investigated by lumbar puncture for definite diagnosis.
Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1–K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 × 107m−1, as determined by surface plasmon resonance, a value close to that (1.6 × 107m−1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.
In a haematology ward, Candida parapsilosis was found in blood cultures from 4 patients within a month. As C. parapsilosis is known to have a restricted genetic diversity, a combined methodological approach was adopted to establish a possible epidemiological relationship among the isolates (n = 9). Multilocus sequence typing and random amplified polymorphic DNA analysis suggested a clonal origin of the isolates. The clonal origin was confirmed by microsatellite analysis, a method that displayed the highest discriminatory level and readily differentiated cluster isolates from 2 epidemiologically unrelated strains of C. parapsilosis. The use of novel methods of genotyping such as microsatellite analysis will facilitate epidemiological investigations of potential clonal outbreaks of fungaemia.
Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.
Pathogenic bacteria often produce potent proteases capable of destroying host tissue thereby providing the bacteria with tools for spreading and nutrient access. This thesis describes how group A, C and G streptococci can acquire surface bound protease activity through an alternative mechanism, namely by binding and activation of the human protease precursor plasminogen.
Group A streptococci of certain serotypes (M33, M41, M52, M53 and M56) were found to efficiently bind plasminogen. Similar to a previously described protein (PAM) expressed by a M53 strain, the plasminogen-binding surface proteins of the other four serotypes belonged to the M protein family, known to contain major virulence factors of group A streptococci. In addition, a subset of group C and G streptococci were shown to bind plasminogen through M-like proteins.
In binding experiments with recombinantly produced fragments and a synthetic pepide we located the plasminogen-binding site of protein PAM to a 29 amino acid region containing a twice-repeated sequence. Two lysine residues within this sequence appeared to be critical for the interaction with plasminogen.
The major binding site for PAM to plasminogen was localised to kringle two of human plasminogen. PAM reacted poorly with plasminogen from some other species, including rheusus plasminogen which only differs from the human form in two positions.
PAM-expressing bacteria grown in human plasma acquired surface associated plasmin activiy in spite of the presence of physiological plasmin inhibitors. A chimerical M-like protein, harbouring the plasminogen-binding motif of PAM, transferred this ability to another streptococcal strain. Inactivation of the streptokinase gene abolished surface plasmin acquisition whereas addition of exogenous streptokinase overcame this block.
The purpose of this study was to assess the seroprevalence of C6 ELISA antibodies in healthy blood donors in Kalmar County, Sweden, in relation to age, sex and time of year (peak season vs off season). In addition, we wanted to assess serological status over time in a group of C6 ELISA seropositive blood donors. Sera were collected from 273 (131 women, 142 men) blood donors in autumn 2011 and 300 (144 women, 156 men) in winter 2014. All sera were analysed in the C6 ELISA and the results were interpreted according to the manufacturer's instructions. The seroprevalence was 22% (females 16%, males 28%) in 2011 and 24% (females 15%, males 33%) in 2014. The seroprevalence was significantly higher in males and increased with age. The highest seroprevalence was observed among elderly men, 60-70 years old (46% in 2011 and 52% in 2014). No significant difference was detected in seropositivity between the samples collected in winter and autumn. All (34/34) seropositive blood donors followed over time remained seropositive at follow-up after 22-29 months. C6 ELISA seroprevalence in healthy blood donors is high in Kalmar County, thereby reducing the specificity of a positive test result regarding the clinical diagnosis of Lyme borreliosis (LB). Although C6 seroprevalence appears not to be affected by seasonal sample time, it varies greatly with age and sex. A careful evaluation of pre-test probability is therefore of the utmost importance in the clinical diagnosis of LB, especially in elderly men. We suggest that colleagues in other endemic regions also consider initiating similar evaluations to optimize the laboratory and clinical diagnosis of LB in relation to age and sex.
