Unusual phage pattern of Salmonella Typhimurium isolated from Swedish patients and Italian salami.
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Adolescent↗
Biomedical subjects
S Löfdahl
Publications and source records attributed to S Löfdahl.
Diversity and evolution of the class A chromosomal beta-lactamase gene in Klebsiella pneumoniae.
We investigated the diversity of the chromosomal class A beta-lactamase gene in Klebsiella pneumoniae in order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified in K. pneumoniae and of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for other K. pneumoniae beta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genes gyrA, coding for subunit A of gyrase, and mdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome of K. pneumoniae.
Amino Acid Sequence↗
A one-year study of foodborne illnesses in the municipality of Uppsala, Sweden.
Surveillance was enhanced and a retrospective interview study performed in 1998-99 to determine incidence, causes, and costs of foodborne illnesses in Uppsala, Sweden. Sixty-eight percent of the detected foodborne illness incidents were single cases, and 32% were outbreaks. Most (85%) of the incidents came to the attention of the municipal authorities through telephone calls from affected persons. Calicivirus, Campylobacter spp., and Staphyloccocus aureus were the most common etiological agents; meat, meat products, and mixed dishes were the most implicated food categories. The incidence of foodborne illness was estimated to be 38 cases per 1,000 inhabitants per year. The estimated average costs per illness were 2,164 Swedish Krona (SEK) ($246) to society and 500 SEK ($57) to the patient. The annual cost of foodborne illnesses in Sweden was estimated to be 1,082 million SEK ($123 million).
Adolescent↗
Enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a Swedish clinic for infectious diseases.
A 1-year prospective study was conducted to identify enteropathogens in adults with diarrhea (n=851) and in healthy control subjects (n=203) by use of conventional laboratory methods. Virulence factor genes for diarrheagenic Escherichia coli were detected by polymerase chain reaction. Enteropathogens were identified in 56% of patients and 16% of control subjects. The isolation rate was 65% for patients with symptoms for <1 week and for travelers; >1 pathogen was found in 11% of patients. The most frequent enteropathogens were Campylobacter (13% of patients), Clostridium difficile (13%), enterotoxigenic Escherichia coli (8%), Salmonella (7%), Shigella (4%), Blastocystis hominis (4%), calicivirus (3%), rotavirus (3%), enteroaggregative E. coli (2%), Aeromonas (2%), Giardia intestinalis (2%), Cryptosporidium (2%), and astrovirus (2%). Less frequently isolated (< or =1% of patients) were verotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, Entamoeba histolytica/Entamoeba dispar, microsporidia, and adenovirus. Fifty percent of the patients were hospitalized, and 43% needed intravenous fluids. The median duration of diarrhea was 14 days. Clinical features were not helpful for predicting the etiology of diarrhea.
Adolescent↗
[Pneumocystis carinii is still a dangerous opportunist. The infection is continuously a threat to immunocompromised patients].
Despite recent decrease in the incidence of Pneumocystis carinii pneumonia (PCP) among patients infected with HIV (human immunodeficiency virus), PCP remains a threat to other categories of immunocompromised patients. The article provides an outline of recent, mainly molecular genetic, findings in P. carinii research, including its new classification as a primitive fungus, host specificity and verified de novo infection in HIV-infected subjects. As the pathogen still defies propagation in vitro, laboratory diagnosis is dependent on microscopic demonstration of the organism. Diagnostic specificity can be enhanced by generating specific PCR (polymerase chain reaction) products which can be sequenced for genotyping. Findings in animal studies and epidemiological observations (e.g., in outbreaks of PCP among immunocompromised hospital patients), suggest transmission of PCP infection to be airborne. Genetic methods have been used to study the mode of P. carinii transmission. Nucleic acids of the human form of P. carinii (P. carinii f. sp. hominis) have been detected in the air of hospital wards, indicating susceptible patients to be at risk. By contrast, findings obtained with the same methods in studies of person-to-person transmission of P. carinii among clustered cases of PCP in hospitals suggest infection to be environmentally acquired. Thus, many questions remain to be answered regarding the occurrence and transmission of P. carinii infection.
AIDS-Related Opportunistic Infections↗
Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease.
On centisome 7, Salmonella spp. contain a large region not present in the corresponding region of Escherichia coli. This region is flanked by sequences with significant homology to the E. coli tRNA gene aspV and the hypothetical E. coli open reading frame yafV. The locus consists of a mosaic of differentially acquired inserts forming a dynamic cs7 region of horizontally transferred inserts. Salmonella enterica subspecies I, responsible for most Salmonella infections in warm-blooded animals, carries a fimbrial gene cluster (saf) in this region as well as a regulatory gene (sinR). These genes are flanked by inverted repeats and are inserted in another laterally transferred region present in most members of Salmonella spp. encoding a putative invasin (pagN ). S. enterica subspecies I serovar Typhi, the Salmonella serovar that causes the most severe form of human salmonellosis, contains an additional insert of at least 8 kb in the sinR-pagN intergenic region harbouring a novel fimbrial operon (tcf ) similar to the coo operon encoding the CS1 fimbrial adhesin expressed by human-specific enterotoxigenic E. coli. It is suggested that the multiple insertions of fimbrial genes that have occurred in the cs7 region have contributed to phylogenetic diversity and host adaptation of Salmonella spp.
Chromosome Mapping↗
Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes.
Enterobacteriaceae were found in high numbers after storage at 7 degrees C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat. Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled. One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30 degrees C for 3d) or Violet red bile glucose (VRBG) agar (37 degrees C for 1d). In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered. On fish, the most frequently found species were R. aquatilis, and in milk, the dominating species were S. liquefaciens, H. alvei and R. aquatilis. One to three isolates of Citrobacter freundii were found in all three food categories. Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin (lt) was indicated in one fish isolate of R. aquatilis whereas heat-stable toxin genes (s.t.) were indicated in four H. alvei isolates, two originating from fish and two from minced meat. Positive PCR-reaction for vero cytotoxin genes were found in one H. alvei strain originating from fish (vt1), in two S. liquefaciens strains from minced meat (vt2), and in a C. freundii reference strain. One of the st-positive H. alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E. coli.
Animals↗
An allelic variant of the chromosomal gene for class A beta-lactamase K2, specific for Klebsiella pneumoniae, is the ancestor of SHV-1.
Fecal Klebsiella isolates from neonates in 22 Swedish special care units were examined by a PCR we developed for detection of the SHV-1 beta-lactamase gene. All 105 K. pneumoniae isolates and all 11 K. pneumoniae reference strains (including the K. pneumoniae subsp. pneumoniae, ozaenae, and rhinoscleromatis type strains) tested were positive, whereas all 67 K. oxytoca isolates and the K. oxytoca, K. planticola, and K. terrigena type strains tested were negative. Resistance to beta-lactams in K. pneumoniae was not transferable by conjugation, and the beta-lactamase gene was never found on a plasmid. Southern blot analysis showed that the gene had a defined chromosomal location. Isoelectric focusing and sequencing of 231-bp PCR amplicons from different isolates revealed many variants of the enzyme, with the two main groups being SHV-1 like (pI 7.6; 68 isolates) and LEN-1 like (pI 7.1; 14 isolates). Clavulanic acid markedly reduced the MICs of ampicillin for all the K. pneumoniae isolates tested. This fact, MIC profiles (penicillin rather than cephalosporin resistance), pIs, and sequence data showed that the chromosomal beta-lactamase of K. pneumoniae is a class A, group 2 enzyme distinct from the chromosomal AmpC enzymes found in several other gram-negative bacteria and from the chromosomal beta-lactamase K1 of K. oxytoca. We propose that the chromosomal beta-lactamase of K. pneumoniae be designated K2 and suggest that an allelic pI 7.6 variant of this enzyme is the ancestor of the SHV family of plasmid-mediated beta-lactamases.
Alleles↗
Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction.
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Base Sequence↗
A rapid and simple nested PCR assay for the detection of Pneumocystis carinii in sputum samples.
Detection of Pneumocystis carinii by the polymerase chain reaction (PCR), based on the thymidylate synthase (TS) gene of rat P. carinii, is a specific and sensitive method for the detection of the parasite in respiratory samples. However, the use of the method is limited by a laborious phenol-chloroform DNA extraction method and an expensive and time-consuming hybridization procedure. For routine clinical samples, DNA preparation can be simplified and hybridization substituted by a nested PCR technique. Such a modified PCR procedure, based on the TS gene of P. carinii, was evaluated on 190 induced sputum samples from 50 immunosuppressed patients, infected with human immunodeficiency virus (HIV), with and without symptoms of P. carinii pneumonia (PCP). The PCR assay, preceded by a rapid DNA preparation (Wizard DNA Clean-up), detected P. carinii-DNA in 13/15 sputa containing parasites as seen by microscopy using immunocytochemical (IFL) staining, and in 10 additional sputum samples lacking demonstrable parasites by microscopy. These samples are to be considered as 'true' positives, since all but 2 were from patients, who developed a PCP within 1 year. We conclude that the nested PCR assay is more sensitive than IFL for the detection of P. carinii in AIDS patients, prior to the debut of PCP symptoms.
Animals↗
Detection of Candida albicans DNA in serum by polymerase chain reaction.
Polymerase chain reaction (PCR) was applied in order to improve the diagnosis of disseminated candidosis. A nested PCR technique with 2 primer pairs was used to increase the sensitivity. We were able to detect Candida DNA in serum and tissue samples from experimentally infected mice as well as in serum samples from candidemia patients and patients with deep-seated Candida infection. Our PCR could detect as little as 1 pg Candida albicans DNA. The PCR method was more sensitive than culture in both the mouse experiments and the patients with deep candidosis (5/7 were PCR positive and 0/7 blood culture positive), and of similar sensitivity in candidemia patients (11/17 were 15/17 blood culture positive). The relatively short processing time of PCR, when compared with culturing, its sensitivity, as well as the possibility of using serum samples for analysis, all helped improve the diagnostics in deep-seated candidosis and candidemia.
Adult↗
Use of a DNA hybridization method to verify results of screening for methicillin resistance in staphylococci.
Tests were performed by the disk diffusion method, agar dilution method and the E test to determine the susceptibility to methicillin and oxacillin of clinical isolates and control strains of Staphylococcus aureus (n = 106) and coagulase-negative species (n = 131). Results were compared with those of a dot blot DNA hybridization test, in which the mecA gene was detected using an oligonucleotide probe selected from the mecA gene. Among the Staphylococcus aureus strains the mecA gene was found in all but two strains inhibited by > or = 8 mg/l of methicillin and all but two strains inhibited by > or = 4 mg/l of oxacillin. A disk test using either 1 microgram oxacillin or 10 micrograms methicillin and a tentative resistance breakpoint of < or = 10 mm gave the best agreement with the hybridization test. For coagulase-negative staphylococci 34 of 35 strains inhibited by > or = 8 mg/l methicillin hybridized with the probe as well as 58 of 82 strains inhibited by 1-4 mg/l; 93 of 97 strains inhibited by > or = 0.5 mg/l oxacillin were also positive in the probe test. Using the 1 microgram oxacillin disk and a resistance breakpoint of < or = 10 mm good agreement was obtained between results of the disk diffusion and DNA hybridization tests. It is suggested that this genotypic method for detection of methicillin resistance is used as a reference method for routine methods.
Base Sequence↗
Detecting methicillin-resistant Staphylococcus epidermidis--disc diffusion, broth breakpoint or polymerase chain reaction?
Growth conditions are important for the expression of resistance to methicillin among staphylococci. Consequently a phenotypic susceptibility test has to be chosen carefully to avoid false susceptible results. In this study we wanted to devise rapid and simple phenotypic tests whose results completely correlate with the presence of the methicillin resistance gene, mecA. A simplified polymerase chain reaction (PCR) method not needing separate DNA extraction from the tested bacteria was used to amplify a 449 bp region of the mecA gene. One hundred and ten strains of S. epidermidis were tested. The results were in complete agreement with those from a broth tube breakpoint test, known to identify more strains as resistant than does the method recommended by NCCLS. In disc diffusion test it was possible to clearly distinguish resistant from susceptible strains by using discs containing oxacillin, cephalexin and cephradine. A 5 micrograms cephradine disc was further analysed by testing another 441 consecutive clinical isolates of staphylococci. All resistant coagulase-negative staphylococci grew out to the edge of this disc, whereas susceptible strains showed an inhibition zone at least 10 mm in diameter. The 5 micrograms cephradine disc is recommended for routine work. The PCR method and broth tube breakpoint test are both reliable reference methods.
Base Sequence↗
Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction.
A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii.
Base Sequence↗
Comparison of the amino acid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains.
The surface (S) genes of 12 hepatitis B viruses (HBVs) encoding nine different serotypes of hepatitis B surface antigen (HBsAg) were amplified by the polymerase chain reaction and sequenced. These represented the eight strains of HBV, P1 to P8, defined at an international workshop on HBsAg subtypes in Paris in 1975, and the adrq- subtype. The S genes from additional HBV strains, one ayw4, one adw4 and one ayw1, of sub-Saharan African origin, were also sequenced. The relationship of these 12 new S gene sequences to those of the 20 published previously was investigated by constructing a phylogenetic tree, which confirmed a previous classification into four groups, designated A to D, based on 18 complete HBV genomes. When relating our sequenced S genes to these genomic groups, ayw1 of African origin and P6 (adw2) were both allocated to group A, the reference P1 (ayw1 of Vietnamese origin) was allocated to group B, P5 (ayr), P8 (adr) and adrq- were all related to group C, and P2 (ayw2) and P3 (ayw3) could both be allocated to group D. Interestingly, the S genes of w4 serotype viruses, i.e. P4 (ayw4) and P7 (adw4q-), differed by 4% or more from both previous groups and from each other, suggesting their classification into two new groups, for which the designations E and F are proposed. Genomes specifying ayw were also found in groups A and B; previously sequenced genomes specifying the ayw subtype have all been confined to group D. There were indications that the epitope for subdeterminants of w resided at amino acid positions 125 to 127. Thus, at positions 125 and 127, ayw1, ayw2 and adw2 had T and P residues, respectively, whereas M and T residues were at the corresponding positions of ayw3. Both ayw4 and adw4 had L at residue 127, and all strains expressing r, apart from P5, had an I instead of a T residue at position 126.
Amino Acid Sequence↗
Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis.
Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast.
Amino Acid Sequence↗
DNA hybridization for diagnosis of pertussis.
The aim of the present study was to evaluate a mixed phase DNA hybridization assay for detection of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal aspirates from patients with suspected pertussis. Among 179 consecutive patients with own or parental suspicion of pertussis, the diagnosis was confirmed in 103 patients by serology and in 52/103 (50%) cases also by culture. The remaining 76 patients served as nonpertussis controls. Direct hybridization was positive in 38% samples with serology as reference method, a non-significant difference to the 50% sensitivity for culture. Preculture of samples on membranes for 24, 48 and 72 h gave a significantly higher sensitivity only with 72 h preculture, 69% vs 50% (P = 0.007). The 72h preculture gave, however, also a significant decrease of specificity, 87% vs 100% for routine culture (P = 0.001) and is not a more rapid diagnostic method. The result shows that rapid diagnosis by DNA hybridization can be achieved in a large proportion of pertussis cases. The presence of smaller numbers of bacteria in samples only positive after preculture indicate that DNA hybridization could be a highly sensitive diagnostic method with further development of more rapid amplification systems.
Bordetella↗
A DNA hybridization test for detection of Bordetella in nasopharyngeal specimens.
A cloned Bam H1 fragment of genomic Bordetella pertussis DNA which recognized a frequently repeated sequence in the genome of B. pertussis was used as a probe in a DNA hybridization assay for the detection of Bordetella. Extensive studies on cross-reactivity were carried out in standardized strains and in cultured swab specimens from patients without suspected pertussis. Hybridizations of cultured clinical specimens from 142 patients with suspected pertussis were in complete agreement with the standard identification methods. The recovery rate of B. pertussis from nasopharyngeal swabs was less than 50%. Therefore the possibility to detect low numbers of B. pertussis in solution (nasopharyngeal aspirates) was investigated. The detection limit of direct hybridization by dot-blot technique was 5 x 10(3)-10(4) B. pertussis. Culturing bacteria on membranes placed on agar plates prior to hybridization showed that the detection limit could be lowered to 10(4), 10(2), and 10(1) cfu after 1, 2 and 3 days' culture, respectively. DNA hybridization under these conditions was found to be sufficiently sensitive and specific for further evaluation in clinical specimens for diagnosis of pertussis.
Bacteriological Techniques↗