[Diagnosis of yersinioses and infections with enterohemorrhagic Escherichia coli].
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Bacteriological Techniques↗
Biomedical subjects
H Karch
Publications and source records attributed to H Karch.
[The immune response in edema disease of weaned piglets measured with a recombinant B subunit of shiga-like toxin II].
An outbreak of edema disease (ED) was monitored in 80 piglets after weaning over a period of 4 weeks. The shedding of Shiga-like toxin-IIe) producing Escherichia coli strains, the serum bactericidal activity (SBA) against SLTEC-IIe, and the antibody response against SLT-IIe were investigated. The antibody response was monitored by utilizing a glutathione-S-transferase (GST) + SLT-IIe B/SUB fusion protein (FRANKE et al., in press) for immunoblot assays. E. coli-strain GO15III (0141:K85ac) was diagnosed as SLT-IIe-producing E. coli by polymerase chain reaction, DNA hybridization and cytotoxicity assays. Maximum excretion of GO15III appeared between days 8 and 15 after weaning. On day 1 after weaning no piglet shed GO15III, while the number increased on day 8 to 53 (66.2%) and on day 15 to 59 (73.8%) of the piglets. 4 week after weaning, GO15III was only isolated from 23 (28.8%) of the piglets. In parallel, serum bactericidal activity against GO15III increased significantly in the sera of 73 (91.2%) piglets, reaching a stable maximum from day 15 on. During the first two weeks after weaning, no piglet yielded detectable SLT-IIe-IgG. However, the number of SLT-IIe-IgG positive piglets increased steadily from day 15. On day 15, 5 (6.2%) piglets were positive in SLT-IIe immunoblot analysis and 29 days after weaning the number increased to 31 (38.8%). These data represent the first serological monitoring of a natural outbreak of edema disease in piglets after weaning by using a recombinant fusion protein (GST+SLT-IIe B/SUB). The recombinant protein proved to be a useful diagnostical tool for monitoring the specific antibody status of piglets.
Animals↗
Highly conserved B-subunit genes of Shiga-like toxin II variants found in Escherichia coli O157 strains.
To determine the degree of heterogeneity among Shiga-like toxin-II (SLT-II)-related toxins present in enterohemorrhagic Escherichia coli O157 strains, slt-IIB-related genes of 15 strains were amplified and sequenced. Of these 15 isolates, six contained only the slt-II-related genes, seven strains harbored slt-II-related genes together with slt-II, and two strains had slt-II-related genes plus slt-I. In strains carrying slt-II-related genes alone or in combination with slt-I, the PCR fragments were directly subjected to Taq cycle sequence analysis. Direct sequencing was not possible with the seven strains possessing both slt-II and slt-II-related genes, since the PCR products contained both genes. In order to allow sequence analysis of these slt-II-related genes, the PCR products were first subjected to restriction enzyme digestion with FokI, which selectively digested slt-IIB. This resulted in an undigested 270-bp fragment consisting of pure slt-II-related genes. Interestingly, comparison of the nucleotide sequences revealed 100% homology of all analyzed 15 slt-IIB-related toxin genes. In addition, the nucleotide sequence of slt-IIB-related toxin genes were identical to slt-IIcB. Our findings indicate that SLT-IIc is a major variant form of SLT-II present in E. coli O157 strains.
Bacterial Toxins↗
The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coli alpha-hemolysin family.
Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype. In this study the hemolytic activity of E. coli O157:H7 strain EDL933 was investigated. Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity. By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E. coli K-12 strains C600 and DH5 alpha became positive for hemolysin production. By transformation of recombinant plasmids carrying a 11.9 kb BamHI fragment and a 5.3 kb SalI fragment of pSK3 hemolytic activity is revealed when transformed in E. coli C600 or DH5 alpha DNA-hybridization of pO157 and subclones with the alpha-hemolysin specific DNA probe was only found under conditions of low stringency. No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes. Our results indicate that a hitherto not described hemolysin belonging to the alpha-hemolysin family is encoded by the 90 kb plasmid of E. coli O157 strains.
Base Sequence↗
Differentiation in virulence patterns of Escherichia coli possessing eae genes.
In this study 98 Escherichia coli strains which belonged to traditional enteropathogenic (EPEC) serotypes and 82 enterohemorrhagic E. coli (EHEC) strains were screened by polymerase chain reaction (PCR) for the presence of E. coli-attaching and -effacing (eae) genes. These strains were also hybridized with the enteropathogenic adherence factor (EAF) probe and examined in the fluorescence actin staining (FAS) test. The results obtained from the individual strains demonstrated that all 26 class I EPEC with localized adherence to HEp-2 cells carried EAF and eae genes. In contrast, of 72 EPEC strains with no or diffuse adherence only 1 strain was EAF positive and 6 strains had eae. Of 82 EHEC strains a total of 75 carried eae sequences. Of considerable interest, 15 of 21 E. coli strains that lost their slt genes during subcultivation were found to be eae positive. As controls a total of 53 enterotoxigenic and enteroinvasive E. coli, and 125 E. coli strains from the normal flora were investigated and all displayed negative results in the eae-PCR. From the 201 strains comprising classical EPEC serotypes, EHEC and E. coli with lost slt genes, a total of 126 displayed a positive FAS test and 122 reacted in the eae-PCR. Only 4 strains were FAS test positive but eae-PCR negative. Our data indicate that E. coli strains possessing the eae genes are heterogenous with respect to their virulence determinants. Loss of virulence plasmids and phage-encoded slt genes either in the host or during storage may contribute to this heterogeneity. The eae-PCR detected all class I EPEC and 91.5% of the EHEC.(ABSTRACT TRUNCATED AT 250 WORDS)
Adhesins, Bacterial↗
Acute childhood neuroborreliosis with a selective immune response to a low molecular weight protein expressed by Borrelia garinii.
In patients with Lyme borreliosis the early immune response to Borrelia burgdorferi is usually directed against the flagellin (41 kD) antigen. However, the sera of 4 out of 12 children with typical early neuroborreliosis contained antibodies to two low molecular weight proteins of 19 and 23 kD. These antibodies were demonstrated in serological tests using Borrelia garinii as antigen source. B. garinii is the species of B. burgdorferi sensu lato associated with neuroborreliosis. In contrast serological tests using B. burgdorferi sensu stricto, associated with arthritis, or using VS461 strains (B. afzelii), associated with chronic skin disease, were false-negative. Monospecific rabbit sera raised against the 23 kD protein of B. garinii strain W1 detected the 23 kD epitope in B. garinii isolates only. Results of serological tests for Lyme borreliosis may depend on the infecting strain and on the choice of the strain used as antigen source for diagnostic tests. The attending physician should interpret serological test results with care and rely on astute clinical judgement.
Adolescent↗
Haemolytic anaemia in association with Escherichia coli O157 infection in two sisters.
Two sisters, 2 and 5 years of age, suffered from acute haemolytic anaemia occurring after gastroenteritis with Escherichia coli O157. One patient developed clinical signs of severe and acute intravascular haemolysis and sepsis. She received transfusion and antibiotic therapy. The second patient presented with mild to moderate haemolytic symptoms only. None of them developed renal impairment. In serum of both children, elevated titres of short-lived agglutinins were demonstrated in the indirect haemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide from E. coli O157. By immunoblot analysis IgM antibodies against the O157 lipopolysaccharide were demonstrated in the acute phase sera but not in follow up sera taken 2 months after disease. On erythrocyte membranes, adsorption of microbial antigens was detected by use of a pool-immunoglobulin fluorescence test. The immunological status of both patients was normal. Complete recovery from haemolytic disease was observed without further therapy. Microbial antigens attached to the cell surface were assumed to be the pathophysiological cause of E. coli O157 associated haemolytic anaemia in two siblings.
Acute-Phase Reaction↗
An outbreak due to enterohaemorrhagic Escherichia coli O157:H7 in a children day care centre characterized by person-to-person transmission and environmental contamination.
An outbreak of gastrointestinal disease and haemolytic uraemic syndrome caused by Escherichia coli O157:H7 was investigated. The outbreak occurred in a day care centre located in northern Germany in August 1992 and involved 39 children and two adults. Furthermore, four asymptomatic infections were detected among the staff. Initial and secondary cases were reported over a 30-day interval, with cases occurring in three waves. Person-to-person contact and environmental contamination were assumed to be the main mode of transmission. The source of the outbreak has remained unknown but it is likely that primary or secondary contamination of the day care centre's kitchen, too, played a role in the spread of infections. The organisms were isolated from two open packs of deep-frozen stuffed cabbage rolls and turkey scallops in batter, and furthermore from swabs from two kitchen utensils. Of the 39 cases with diarrhoea, three developed a haemolytic uraemic syndrome; one of the latter patients died. In 8 of the cases as well as in four healthy adult employees, E. coli O157:H7 was isolated from stool samples, and in two stool culture-negative cases the presence of IgM antibody to O157 LPS indicated recent infection. The E. coli O157:H7 isolates from the cases and the kitchen were of identical phage type and yielded identical biochemical reactions. All E. coli O157:H7 isolates harboured stable slt-II genes. However, slt-I genes could only be demonstrated in the primary cultures and were lost during subcultivation. This is the largest outbreak caused by enterohaemorrhagic E. coli O157:H7 that has been documented in Germany so far. The high infectivity of the organism which was demonstrated by person-to-person transmission and propagation within certain groups of children stresses the need for strict hygienic measures and early case reporting when such infections occur in susceptible settings like day care centres, nursing homes, or hospitals.
Adult↗
Prevalence of attaching and effacing Escherichia coli in stool samples from patients and controls.
Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) have the ability to cause 'attaching and effacing' (AE) lesions; the genes necessary to cause AE in both of these pathogroups have been identified and termed eae. Using colony hybridization, we screened 237 stool samples from patients with diarrhea, and 237 stool samples from age-matched controls for the presence of E. coli carrying eae. Individual colonies harbouring eae could be recovered from 7 (2.9%) of the patient stools, as well as from 6 (2.5%) of the control stools. All these E. coli isolates were positive in the fluorescence actin staining (FAS) test. In addition, all the samples were also probed for Shiga-like toxin (slt) genes and the EPEC adherence factor (EAF) to evaluate whether testing for eae identified all EHEC and class I EPEC. Of the 7 patient samples harbouring E. coli with eae, 4 had E. coli with eae and slt genes, and 2 had E. coli with eae and EAF sequences. In 2 of the 237 patient stools, E. coli which were eae and EAF negative but slt probe positive could be recovered. These 2 E. coli strains were non-reactive in the FAS test. Of the control samples, none of the E. coli strains, including the 6 samples containing eae positive strains, possessed EAF or slt-sequences. In concrete terms, the similar eae incidence found in both E. coli isolates from patients and controls is currently of limited clinical diagnostic value and more importantly, the eae probe could not identify all slt-harbouring E. coli. On the basis of these results, the use of the eae-probe cannot be recommended in preference to the slt probes for the detection of EHEC.
Adhesins, Bacterial↗
A fatal case of hemorrhagic colitis, thrombocytopenia, and renal failure associated with verocytotoxin-producing, non-O157 Escherichia coli.
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Bacterial Toxins↗
Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome.
The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli (O)157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs--one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.
Animals↗
DNA fingerprinting of Eikenella corrodens by pulsed-field gel electrophoresis.
In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed-field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed-field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15-20 fragment bands that were clearly separated by the following pulsed-field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120 degrees. These conditions enabled us to distinguish 8 individual pulsed-field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same time revealing this pulsed-field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.
Bacterial Outer Membrane Proteins↗
Polymerase chain reaction for the identification of Porphyromonas gingivalis collagenase genes.
Porphyromonas gingivalis has been shown to exhibit genetic diversity possibly resulting in variation of virulence. In the present study a potential virulence factor was targeted for the detection of P. gingivalis. A 548 bp fragment of the collagenase gene (prtC) from Porphyromonas gingivalis ATCC 33277 was amplified by polymerase chain reaction (PCR) using oligonucleotides derived from the middle portion of prtC. From 16 of 21 clinical P. gingivalis strains, a PCR product of similar size to the prtC could be obtained. These 16 P. gingivalis strains were confirmed as positive for prtC using DNA hybridization with a digoxigenin-labeled prtC PCR product as a probe. In 12 of the 16 prtC positive strains, the restriction analysis of the PCR products revealed fragment patterns identical to the known sequence. In the other 4 prtC positive strains, 4 distinct patterns were found. Of these strains, nucleotide sequence analysis of a 400 bp PCR product stretch revealed 79.1%, 83.0%, 84.8 and 89.5% homology with the known nucleotide sequence for this specific region. Sequence analysis of the PCR products from the ATCC 33277 strain demonstrated 93.7% homology. The limit of detection for the PCR was about 100 organisms. None of the other 48 tested strains of 16 bacterial species derived from oral and extraoral infections yielded a PCR product. The PCR was also used for the detection of prtC sequences in dental plaque. Our data indicate that not all P. gingivalis strains have prtC. Nucleotide heterogeneity exists among P. gingivalis with prtC.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Proteins↗
Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker.
To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.
Base Sequence↗
Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids.
The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pK18. The nucleotide sequence of this fragment was determined, and a set of primers was designed to amplify a 397-bp region associated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known genes. However, between bases 701 and 787 of the fragment, an 82.8% homology between the EAF and the insertion sequence IS630 of Shigella sonnei exists. The results of PCR with primers of the EAF sequence demonstrated that all of the 151 EAF probe-positive EPEC strains with localized adherence to HEp-2 cells yielded positive EAF PCR results. In contrast, none of the 277 EAF probe-negative strains reacted to the EAF PCR. In addition, the PCR assay was successfully used to generate vector-free digoxigenin-labeled EAF fragments that gave valid results in colony blot hybridization assays. The EAF PCR appears to be a specific and efficient method for the detection of EPEC strains carrying the EAF plasmids.
Adhesins, Escherichia coli↗
Demonstration of Borrelia burgdorferi DNA in urine samples from healthy humans whose sera contain B. burgdorferi-specific antibodies.
Since the possibility of asymptomatic infection with Borrelia burgdorferi has been suggested by a positive serology found in healthy subjects, we hypothesized that these subjects might excrete borrelial DNA sequences in urine as happens in patients with Lyme borreliosis. We found borrelial sequences by nested PCR in the urine samples from 3 of 13 healthy B. burgdorferi antibody-positive adults but not in urine samples from 79 antibody-negative healthy controls. After therapy with doxycycline, the urine samples were repeatedly negative for B. burgdorferi DNA. We conclude that urinary excretion of borrelial DNA sequences may occur in seropositive healthy subjects during asymptomatic infection. Demonstration of such sequences in urine must be interpreted cautiously and may not necessarily prove a borrelial cause of disease.
Adult↗
[The effect of the pipe material of the drinking water system on the frequency of Legionella in a hospital].
In this paper the prevalence of Legionella in water samples from cold and warm water supply systems made of copper, iron and polyethylene was determined. Water supplied by copper pipes revealed to be nearly free of Legionella (only 2% of probes positive), but water from iron (90%) or polyethylene pipes (65% probes positive) proved to be heavily contaminated. The 82 isolates were identified as Legionella pneumophila, one from serogroup 1, the others from serogroup 4.
Colony Count, Microbial↗
[Infectious causes of diarrhea in Central Europe].
In this paper the most isolated endemic and imported pathogens of diarrhea in Central Europe are demonstrated. Clinical symptoms, diagnosis and therapy and additional hygiene measures are described.
Anti-Bacterial Agents↗