Biomedical subjects

G Kronvall

Publications and source records attributed to G Kronvall.

At least 37 records · Page 2Linked to original sources

PubMed1999

Genetic characterization of resistance to extended-spectrum beta-lactams in Klebsiella oxytoca isolates recovered from patients with septicemia at hospitals in the Stockholm area.

Two beta-lactamase gene regions were characterized by DNA sequencing in eight clinical isolates of Klebsiella oxytoca. The blaOXY-2a region encoded a beta-lactamase nearly identical to OXY-2 (one amino acid residue substituted) and conferred aztreonam and cefuroxime resistance on the K. oxytoca isolates. Overproduction of OXY-2a was caused by a G-to-A substitution of the fifth nucleotide in the -10 consensus sequence of blaOXY-2a. The blaOXY-1a was identified in a susceptible strain, and the OXY-1a enzyme differed from OXY-1 by two amino acid residues.

Amino Acid Sequence↗

PubMed1999

Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type beta-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC beta-lactamase.

Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like beta-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum beta-lactams in E. coli; nevertheless, the phenotype was cryptic in the K. oxytoca donor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 beta-lactamase (blaCMY-5). The blaCMY-5 product was similar to the plasmidic CMY-2 beta-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. blaCMY-5 was followed by the Blc and SugE genes of C. freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C. freundii; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype of blaCMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

Amino Acid Sequence↗

PubMed1999

Epidemiological typing of Streptococcus pneumoniae from various sources in Sweden and India using Box A PCR fingerprinting.

Epidemiological typing of Streptococcus pneumoniae is essential to determine strain relatedness and also to trace resistant clones. The novel Box A PCR assay was used for characterization of S. pneumoniae isolates from two Scandinavian countries and to compare those from India on the Asian continent. In addition, the assay was employed to determine the clonality of 25 pneumococcal strains from an outbreak in a day-care centre in Linköping, Sweden. All 25 showed a unique pattern with 100% homology for 24 of them, thereby establishing the clonal nature of the outbreak. The pneumococcal strains involved in the outbreak belonged to serotype 9 and were resistant to penicillin, with an MIC value of 2 mg/l. Thirty-eight genotypes were obtained when the Box A results were analysed by computer (Molecular Analyst Software with GelCompar). The discriminatory index of the method was D=0.98, which indicates excellent performance. No major segregation of strains from the different geographical locations was observed when a lower level of similarity was used for typing (80%, 13 types). However, at the level chosen for genotyping, 95% (38 genotypes) there was a clear geographical segregation. No correlation between genotype and serotype was seen as strains from a common place of origin were most often of different serotypes. Computer-assisted analysis of the results of Box A PCR typing facilitated the evaluation.

DNA Fingerprinting↗

PubMed1998

Binding to human extracellular matrix by Neisseria meningitidis.

Adhesion of Neisseria meningitidis strains to extracellular matrix (ECM) and purified matrix components was examined. Most strains bound to subendothelial ECM as well as to immobilized fibronectin and types I, III, and V collagen. Strains from healthy carriers adhered significantly better than isolates from patients. The binding site was localized to the central 75-kDa cell-binding domain of the fibronectin molecule. This domain has not been described previously to interact with bacterial structures.

Bacterial Adhesion↗

PubMed1997

Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.

Amino Acid Sequence↗

PubMed1997

Pseudomonas aeruginosa adherence to external auditory canal epithelium.

BACKGROUND: Pseudomonas aeruginosa is an important causative agent for external otitis. The specific bacterium-host reaction has not been investigated. It is therefore unknown whether adhesion of the external otitis strain to the external auditory canal epithelium is increased compared with strains isolated from other infections. DESIGN: A cohort study was designed to outline adhesion of P aeruginosa to the external auditory canal epithelium, cultured in vitro, of the guinea pig. Factors important for pathogenesis were also studied. PATIENTS: Pseudomonas aeruginosa strains from nonhospitalized patients were collected consecutively at the bacteriological laboratories at Karolinska Hospital, Stockholm, and Huddinge Hospital, Huddinge, Sweden. External otitis strains were compared with strains from leg ulcers, urinary tract infections, and cystic fibrosis. METHODS: Adhesion to the external auditory canal epithelium cultured in vitro was measured and compared groupwise with the mean profile of pathogenic factors. RESULTS: Adhesion to the epithelium was significantly increased for external otitis strains. These strains also had a significantly increased deoxyribonuclease production and a significantly decreased production of pyocyanin and alginate. CONCLUSIONS: The significantly increased ability of P aeruginosa, isolated from external otitis, to adhere to external auditory canal epithelium was combined with a significant production of pathogenic factors. The P aeruginosa that causes external otitis could therefore be considered a particular phenotype. The enzyme profile for external otitis strains was similar to that of the control groups except for the strains from cystic fibrosis. Adhesion to guinea pig vs human epithelium must be compared, and the effects of extracellular proteins on adhesion should be studied to further understand how P aeruginosa adheres to the external auditory canal.

Animals↗

PubMed1997

Reports on surveillance of antimicrobial resistance in individual countries.

In preparation for the meeting of the World Health Organization Working Group on Monitoring and Management of Bacterial Resistance to Antimicrobial Agents, representatives of 10 countries were asked to provide brief reports on the status of surveillance in their countries. Some gave extensive information on the methods used to test susceptibility of nosocomial pathogens to a variety of antibiotics; some described in detail the network of reference laboratories available to hospitals and individual clinicians for monitoring, identifying, and testing infectious agents; others chose to describe how their countries deal with the resistance of the most frequently isolated pathogen to a commonly used drug. The following summary of these reports shows the broad range of problems encountered and solutions undertaken by these 10 countries in dealing with the increasingly alarming problem of bacterial resistance to antimicrobial agents.

Algeria↗

PubMed1997

Antimicrobial susceptibility testing in Sweden. II. Species-related zone diameter breakpoints to avoid interpretive errors and guard against unrecognized evolution of resistance.

The Swedish Reference Group for Antibiotics appointed a subcommittee on methodology (SRGA-M) in 1987 to investigate ways of defining interpretive breakpoints for antimicrobial susceptibility testing. The minimum inhibitory concentration (MIC) breakpoints for susceptibility categories are mainly based on pharmacological properties of the antibiotic, and they are, with few exceptions, valid for all species. However, for several species the MIC breakpoints have failed to distinguish strains with reduced susceptibility from normal susceptible strains. Disk diffusion is the routine method for susceptibility testing in Sweden. Studies of distribution of MICs and zone diameters for clinically important bacterial species have resulted in an emphasis on resistance rather than on susceptibility. The SRGA-M chose to place the zone diameter breakpoints close to the native (often susceptible) population of each species or group of related species. Such species-related zone diameter breakpoints used for susceptibility categories no longer correspond to the pharmacological MIC breakpoints, but divide each species into the fully susceptible (native) population and into those isolates/populations that have acquired a resistance mechanism, resulting in high- or low-grade resistance. By this method the risk of reports of false susceptibility is minimized and early detection of the emergence of antibiotic resistance is ensured.

Anti-Bacterial Agents↗

PubMed1996

Allelic variation of the streptokinase gene in beta-hemolytic streptococci group C and G isolates of human origin.

Genetic diversity of the streptokinase gene (sk) from 36 strains of S. equisimilis and 54 strains of group G streptococci was examined. The strains were isolated from patients with various streptococcal disease manifestations and healthy carriers. The region of the gene that corresponds to amino acid residues 174-244, was PCR amplified. The amplified product was subjected to MluI, PvuII, DraI and DdeI digestion. Based on the restriction enzyme digestion patterns nine sk alleles were recognized. There was no correlation between the various sk gene alleles and streptococcal disease manifestations. Three of the nine sk gene alleles, sk4, sk7, and sk8, were detected earlier among group A streptococci. The other six alleles were unique to S. equisimilis and group G streptococci. The most common alleles were sk5, found in 21/90 (23%) and sk10 detected in 43/90 (47%) of the strains. Alleles sk1 and sk2, the most frequent among group A streptococci, were not found among the strains in the present investigation. Thus, it appears that the sk gene has been evolving in line with other species distinguishing features of the streptococci.

Alleles↗

PubMed1995

Streptokinase activity among group A streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations.

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Adolescent↗

PubMed1994

Binding of tissue-type plasminogen activator (t-PA) to Neisseria meningitidis and Haemophilus influenzae.

Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20-46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10,000 receptors per bacterium for t-PA with a Kd value of about 20 nmol l-1. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l-1. t-PA binding could be reduced about 40% by the addition of 10 mmol l-1 of the lysine analogue epsilon-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 mumol l-1 of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.

Aminocaproic Acid↗

PubMed1993

Streptokinase gene polymorphism in group A streptococci isolated from Ethiopian children with various disease manifestations.

Certain variants of streptokinase from group A streptococci have been associated with acute post-streptococcal glomerulonephritis (APSGN). The streptokinase gene (ska) has previously been grouped into nine different polymorphic genotypes of which ska1, ska2, ska6, and ska9 were identified in group A streptococci associated with clinical and experimental APSGN. A total of 53 group A streptococci isolated from Ethiopian children: five from acute rheumatic fever, 18 from APSGN, ten each from tonsillitis, impetigo and healthy carriers, were analyzed for ska gene polymorphism using polymerase chain reaction (PCR) and restriction enzyme analysis. The frequency of the nephritis-associated streptokinase genotypes was 83% among the APSGN isolates and 74% in the non-ASPGN isolates. ska2 was the most commonly found genotype with a frequency of 64% among all isolates, 66% among the APSGN isolates, and 63% among the non-APSGN isolates. ska1 was identified in 13% among all isolates and 17% among the APSGN isolates. Seventeen non-APSGN isolates from Scandinavian countries were studied for comparison and all carried either ska1 or ska2. The other nephritis-associated ska6 and ska9 were not detected among the 53 Ethiopian isolates. ska1 was exclusively associated with serum opacity reaction (SOR) producers. ska2 was evenly distributed among SOR-positive and SOR-negative isolates. The other genotypes were detected only among SOR-negative strains. The findings of the present study showed an even distribution of the nephritis-associated streptokinase gene among group A streptococcal isolates with no correlation to disease pattern. Thus additional factors must also be operative in the pathogenesis of APSGN.

Acute Disease↗

PubMed1993

Prevalence of potential respiratory disease bacteria in children in Ethiopia. Antimicrobial susceptibility of the pathogens and use of antibiotics among the children.

Acute respiratory infections are primary causes of morbidity and mortality in children in developing countries. This project was designed to investigate antimicrobial susceptibility of respiratory tract pathogens isolated from children in rural and city areas, and to contribute to the rational choice of antibiotics for respiratory tract infections in children in Ethiopia. Nasopharynx and throat cultures were taken from all children under five years of age in three study areas representing different levels of contact with health care and accessibility to modern drugs, such as antibiotics. In all, 1126 children were cultured. Haemophilus influenzae and Streptococcus pneumoniae were both found in 85-90% of the children, and beta-haemolytic streptococci group A in 12%. The level of antimicrobial resistance was low. None of the 954 strains of H. influenzae were beta-lactamase producers. Pneumococci were susceptible to penicillin. The use of antibiotics was also low; 11 of 1126 children had antibiotics on the day of culture or the day before. The choice of antibiotics was not limited by resistance, and emphasis could be put on low cost, minimizing adverse drug reactions and ecological impact.

Anti-Bacterial Agents↗

PubMed1993

Distribution of presumptive pathogenicity factors among beta-hemolytic streptococci isolated from Ethiopia.

Beta-hemolytic streptococci are known to bind several mammalian proteins, which are presumed to be important in pathogenicity. The distribution of such binding structures was examined for mouse albumin, human serum IgA, human IgG, human fibrinogen, and human plasminogen. A total of 218 group A beta-hemolytic streptococci (GAS) were studied: 5 isolates from children with acute rheumatic fever (ARF), 18 from acute post-streptococcal glomerulonephritis (APSGN), 57 from tonsillitis, 52 from skin infections, and 86 from healthy carriers. Sixty-eight Streptococcus equisimilis and 20 group G streptococci were also included. Most of the S. equisimilis (60/68) and group G (14/20) were obtained from apparently healthy carriers. The results were evaluated with respect to T type, serum opacity reaction (SOR), site of isolation, and disease type. No direct correlation was detected between the protein-binding structures studied. There was no apparent correlation between any particular protein-binding structure and specific T type. Albumin-binding and IgA-binding activities were inversely correlated among skin and nephritis GAS isolates. A strong correlation was demonstrated between IgA-binding activity and SOR production, while albumin-binding activity correlated with SOR-negative strains. Albumin-binding levels in isolates from ARF, APSGN and tonsillitis were significantly higher than in isolates from healthy carriers (P < 0.001). A higher albumin-binding capacity was shown in skin isolates from APSGN than in isolates from impetigo (P < 0.001).

Animals↗

PubMed1992

A one-year study of streptococcal infections and their complications among Ethiopian children.

Post-streptococcal complications are known to be common among Ethiopian children. Little is known, however, about the epidemiology of beta-haemolytic streptococci in Ethiopia. A total of 816 children were studied during a one-year period: 24 cases of acute rheumatic fever (ARF), 44 chronic rheumatic heart disease (CRHD), 44 acute post streptococcal glomerulonephritis (APSGN), 143 tonsillitis, 55 impetigo, and 506 were apparently healthy children. Both ARF and APSGN occurred throughout the year with two peaks during the rainy and cold seasons. The female:male ratio among ARF patients was 1.4:1 and 1:1.9 among APSGN. The monthly carrier rate of beta-haemolytic streptococci group A varied from 7.5-39%, average being 17%. T type 2 was the most frequent serotype. Marked seasonal fluctuations were noted in the distribution of serogroups among apparently healthy children. Beta-haemolytic streptococci group A dominated during the hot and humid months of February-May. Strains were susceptible to commonly used antibiotics, except for tetracycline.

Carrier State↗

PubMed1992

Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin.

Forty-two strains of Neisseria meningitidis and 17 of Neisseria gonorrhoeae were tested for their ability to interact with 125I-labeled Glu-plasminogen. All strains tested reacted substantially with plasminogen, resulting in uptake values of 20%-48%. Scatchard analysis with selected N. meningitidis strains demonstrated a dual-phase receptor interaction, one more avid receptor with a Kd of 50 nM and 3000-6000 receptors per bacterium and a second receptor with a Kd of 200 nM and 10,000-20,000 receptors per bacterium. Plasminogen uptake could be completely eliminated by low concentrations of epsilon-aminocaproic acid, suggesting that the lysine binding sites on the plasminogen molecule are involved in the receptor-ligand interaction. The binding of plasminogen to the bacterial receptor facilitates the tissue-type plasminogen activator-mediated conversion to Glu-plasmin, which also modifies itself to the Lys form. Receptor-associated plasmin is enzymatically active, monitored as a breakdown of the chromogenic substrate S-2251, and retains its activity in the presence of naturally occurring inhibitors in plasma.

Aprotinin↗

PubMed1992

Extended spectrum beta-lactamase from Klebsiella oxytoca, not belonging to the TEM or SHV family.

In clinical isolates of Klebsiella oxytoca resistance to cefuroxime and aztreonam was mediated by a beta-lactamase, designated KH, (pI 5.25) which could be transferred into Escherichia coli by electroporation, but not by conjugation. The transformants produced two enzymes with pIs 5.25 and 8.4 respectively, and showed resistance to cefuroxime, aztreonam, cefotaxime and ceftazidime. Substrate and inhibition profiles indicated that KH beta-lactamase was different from TEM- or SHV-like enzymes, but similar to chromosomal K1 beta-lactamase. The enzyme profile with pI 8.4 was similar to the enzyme from the recipient and showed elevated activity in transformants. The plasmid profiles of the transformants were different from those of their donors. However, a plasmid fragment of the K. oxytoca isolate KH11 hybridized with a plasmid ranging in size from 4.8 to 7.8 kilobases in all the transformants and most of the donors. Gene probes encoding TEM-1 or SHV-1 did not hybridize with plasmid DNA from the K. oxytoca isolates. Furthermore, a probe of the ampC gene did not hybridize with the plasmid but to DNA fragments of the same size in whole cell DNA preparations from the E. coli XAC recipient and the TKH11 transformants. This indicates that no gross rearrangements in the chromosomal beta-lactamase gene region had occurred in the transformants which could explain the increased expression of the pI 8.4 beta-lactamase.

Aztreonam↗

PubMed1992

Two types of receptors for human plasminogen on group G streptococci.

To investigate the nature of plasminogen binding to streptococci, strains selected for high reactivity with human plasminogen were examined for binding pattern against a panel of plasminogen fragments. The strains included human isolates of groups A, C and G as well as bovine isolates of group G. All strains reacted substantially with the plasminogen fragment kringle 1-3. Using the miniplasminogen fragment (kringle 5 and the B chain) a small but reproducible uptake was detected for human group G strains but not for group A or C strains. The group G strains of bovine origin on the other hand demonstrated high uptake of miniplasminogen, suggesting the possibility of an alternative plasminogen receptor for this species. This interpretation was supported by blocking experiments with the lysine analogue EACA where low concentrations (1 mM) completely blocked plasminogen binding to human streptococci, whereas a 100-fold higher concentration was needed for bovine group G strains. Scatchard plots with human isolates resulted in straight lines and Kd values were generally in the range of 20-80 nM. The number of receptors was estimated to be 45,000 for a selected group A strain and about 10,000 for the selected group C and G strains. Scatchard analysis with bovine group G isolates on the other hand revealed a two phase interaction, supporting the assumption of two different receptor structures on these strains. Kd for the first phase was estimated to be about 20 nM (10,000-20,000 receptors per bacterium), which was similar to the human strains, whereas the second phase was in the range of 400-500 nM (50,000 and 150,000 receptors per bacterium with two selected strains). Scatchard plots with the miniplasminogen fragment as ligand mimicked the phase two reaction with plasminogen, supporting the concept that this reaction represents a new and not previously described receptor. Both the receptor reacting with the kringle 1-3 portion and the one reacting with the miniplasminogen portion bound plasmin and plasminogen with similar affinity.

Aprotinin↗