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Objective: Wound healing can be delayed by the presence of colonising bacteria, and in polymicrobial wounds they may act synergistically to the further detriment of wound healing. In this pilot investigation, biopsy and swab samples were obtained as part of skin-graft operations performed on a chronic venous leg ulcer in order to study the spatial microbial diversity and to compare standard bacteriological and molecular biological techniques., Method: The wound was sampled before excision, and sampling was undertaken at multiple locations across the wound. Swab samples and biopsies were subjected to culture analysis and 16S rRNA polymerase chain reaction (PCR), and to denaturing gradient gel electrophoresis (DGGE)., Results: Within the wound samples, DGGE identified the major wound microflora components and established the extent of local differences in bacterial diversity., Conclusion: This ongoing investigation has verified DGGE as a powerful tool for elucidating the clinical microbiology of a chronic disease state. It also suggests that skin graft operations are a novel way of obtaining multiple samples for in vivo bacteriology and for establishing the spatial distribution of bacteria in the complex micro-environment of chronic wounds.

Streptococcus pneumoniae (pneumococcus) is a significant cause of morbidity and mortality in infants and elderly people. Pneumococcal strains possess a polysaccharide capsule, and 90 different serotypes have been identified. The pneumococcal ELISA suggested by World Health Organization (WHO) guidelines is based on capsular serotypes included in the 23-valent polysaccharide vaccine (Pneumovax((R))). Pneumococcal antigens were developed and applied in an ELISA, which elicited a low baseline cross-reaction with the common cell wall polysaccharide (C-Ps). The aim of this study was to develop an ELISA using type-specific antigens from S. pneumoniae strains with serotypes of own selection in order to quantify type-specific pneumococcal antibodies in human serum.

Background: In contrast to most populations worldwide, the incidence of gastric cancer increases among Inuit in Greenland. Contributing factors to this increase are unknown, but Helicobacter pylori may be involved. However, little is known regarding the epidemiology of H. pylori in Arctic communities. With the aim of determining age-specific prevalence, risk factors, and association with clinical conditions of H. pylori infection, we carried out a population-based study of H. pylori in Sisimiut, the second biggest town of Greenland., Materials and Methods: A population-based sample of 685 persons had serum drawn that was analyzed for H. pylori IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Risk factors analyses were carried out using multivariate logistic regression models., Results: The seroprevalence was lowest among children aged 0-4 years (6%), but increased rapidly thereafter. In persons aged 15-87 years the seroprevalence had stabilized around 58%. Total number of children in household, number of older, but not younger, siblings and narrow age gap to closest older sibling were associated with H. pylori seropositivity. In contrast, number of adults in household and socioeconomic status did not influence serostatus., Conclusions: The age-specific prevalence pattern in Greenland is intermediate between that of developing and developed countries. The risk factor pattern indicates crowding and older siblings in particular to be key elements in risk of infection.

Infectious gastroenteritis is one of the most common diseases in young children. To clarify the infectious etiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-control study. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, and their parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected by enzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, thermotolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxin-producing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinvasive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence gene probes and serotyping. Parasites were detected by microscopy. Overall, a potential pathogen was found in 54% of cases. More cases than controls were infected with rotavirus, Salmonella, norovirus, adenovirus, Campylobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, although common, was not associated with illness. The single most important cause of diarrhea was rotavirus, which points toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapovirus were also major etiologies. Salmonella sp. was the most common bacterial pathogen, followed by Campylobacter, STEC, Yersinia, and classical EPEC strains. A/EEC not belonging to the classical EPEC serotypes was not associated with diarrhea, underscoring the importance of serotyping for the definition of EPEC.

An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.

The analysis of Serratia liquefaciens MG1 'luxAB insertion mutants that are responsive to N-butanoyl-L-homoserine lactone revealed that expression of lipB is controlled by the swr quorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.

Moraxella canis was isolated from an infected foot ulcer in a patient suffering from diabetes mellitus with neuropathy. Bacteriological findings and 16S rDNA data are presented.

In a human gastric biopsy specimen, 30% of adhering Helicobacter pylori strain AF4 (cagA and VacA positive) was associated with adhesion pedestals. In an AGS cell assay, only a few percent of this type I strain was found to be associated with adhesion pedestals. Nevertheless, a larger proportion of the type I strain was found to invade AGS cells (P < 0.03) and to attach with depressions in the AGS cell membrane (P < 0.03) than a type II strain (cagA and VacA negative). Incubation of AGS cells and H. pylori without adding fetal calf serum (FCS) to the culture medium increased actin accumulations (FITC-phalloidin stained) beneath adhering H. pylori, and decreased H. pylori invasion of AGS cells significantly (P < 0.01). However, no increase in the number of adhesion pedestals was observed by electron microscopy. Proteinase K treatment of FCS eliminated the H. pylori invasion promoting effect (P < 0.01). Our results suggest differences in the ability of H. pylori to induce adhesion pedestals in human gastric epithelial cells and in AGS cells, but a correlation between adhesion pedestal formation in vivo and H. pylori invasion in vitro can be speculated. In addition, H. pylori invasion into AGS cells was found to be mediated by proteins in FCS.

Background: Helicobacter pylori plays an important role in peptic ulcer disease, although not all H. pylori-infected persons will develop a peptic ulcer. Currently, H. pylori strains cannot be divided into commensals and pathogens., Methods: Fifty H. pylori strains were cultured from patients divided into five groups on the basis of upper endoscopic findings: gastric ulcer, duodenal ulcer, gastritis, esophagitis, or normal. The ultrastructural adherence pattern in vivo, autoagglutination, hemagglutination, adhesion to human gastric adenocarcinoma (AGS) cells, and the lipopolysaccharide (LPS) profile of H. pylori strains were recorded; randomly amplified polymorphic DNA (RAPD) and urease gene typing were performed and correlated with diagnostic groups., Results: Electron micrographs showed that H. pylori strains from patients with gastric ulcers adhered more frequently through filamentous strands and were less frequently found free in mucus than any other diagnostic group (P < 0.0001). Neither median hemagglutination titer nor median adhesion capacity to a human gastric adenocarcinoma cell line was related to endoscopic findings. Nevertheless, H. pylori strains from patients with gastric ulcers were more prone to autoagglutinate than were strains from the other diagnostic groups (P = 0.03). H. pylori strains from gastric ulcer patients were found to be more homogeneous, as determined by RAPD and urease gene typing, than strains from the other diagnostic groups (P < 0.01). In addition, a positive correlation was found between a patient's age and the adhesion to AGS cells of the patient's H. pylori strain (P = 0.006)., Conclusion: A combination of an H. pylori autoagglutination test, RAPD, and urease gene typing may be useful in separating gastric ulcer-related strains from duodenal ulcer-related and non-ulcer dyspepsia-related strains.

A model of ascending unobstructed urinary tract infection (UTI) in mice was developed to study the significance of the antibiotic concentration in urine, serum, and kidney tissue for efficacy of treatment of UTI in general and pyelonephritis in particular. Outbred Ssc-CF1 female mice were used throughout the study, and Escherichia coli was used as the pathogen. The virulence of 11 uropathogenic E. coli isolates and 1 nonpathogenic laboratory E. coli strain was examined. Strain C175-94 achieved the highest counts in the kidneys, and this strain was subsequently used as the infecting organism. The model gave reproducible bladder infections, i.e., bacteria were recovered from 22 of 23 control mice after 3 days, and histological examination of kidney tissue showed that of 14 infected kidneys, 7 (50%) showed major histological changes, whereas 3 of 36 uninfected kidneys showed major histological changes (P = 0.018). Once the model was established, the efficacies of different doses of cefuroxime and gentamicin, corresponding to active concentrations in urine only or in urine, serum, and kidney tissue simultaneously, were examined. All cefuroxime doses resulted in significantly lower counts in urine than control treatments, but the dose which produced concentrations of cefuroxime only in urine and not in serum or kidney tissue had no effect on kidney infection. Even low doses of gentamicin (0.05 mg/mouse) resulted in concentrations in renal tissue for prolonged times due to accumulation. All gentamicin doses had a significant effect (compared to the effect of the control treatment) on bacterial counts in urine and kidneys. The antibiotic effect on bacterial counts in bladders was negligible for unknown reasons. Use of the mouse UTI model is feasible for study of the effect of an antibiotic in the urinary system, although the missing antibacterial effect in the bladder needs further evaluation.

Adhesion mediated by fimbriae is thought to play an important role in the pathogenesis of urinary tract infections (UTI) by Escherichia coli. The majority of clinical isolates of E. coli from UTI are able to express type 1 fimbriae. However, the importance of these fimbriae as a virulence factor has been controversial. To investigate the expression of type 1 fimbriae in vivo during UTI, mice were transurethrally infected with uropathogenic E. coli C175-94 and type 1 fimbrial expression was determined directly by two independent methods at 2 h, 1 d and 3 d after infection. By use of an assay combining in situ rRNA hybridization and immunofluorescence, all bacterial cells detected in urine, bladders and kidneys from mice sacrificed 1 and 3 d after onset of infection were found to express type 1 fimbriae. In contrast, the majority of cells in the suspension used for infection of mice and specimens from mice sacrificed 2 h after inoculation were found to be non-fimbriated. Similar results were obtained with a PCR assay revealing the orientation of the invertible promoter driving the transcription of type 1 fimbrial genes. Whilst the promoter in both ON and OFF positions could be amplified from the suspension used for infection and specimens from mice sacrificed 2 h after inoculation, at 1 and 3 d after onset of infection only the promoter in the ON orientation could be amplified. These results show that introduction of E. coli C175-94 into the mouse urinary tract leads to markedly enhanced expression of type 1 fimbriae.

Microbial typing is a useful tool in clinical epidemiology for defining the source and route of infection, for studying the persistence and reinfection rates, clonal selection in the host and bacterial evolution. Phenotypic methods such as biotyping, serotyping and hemagglutinin typing have little discriminatory power compared to genotypic methods concerning the typing of Helicobacter pylori. Therefore great efforts have been made to establish useful molecular typing methods. In this context, the most frequently used genotypic methods are described based on our own experience and the literature: (1) restriction endonuclease analysis, (2) endonuclease analysis using pulsed-field gel electrophoresis, (3) ribotyping, (4) polymerase chain reaction (using either random primers or repetitive DNA sequence primers), and (5) polymerase chain reaction-restriction fragment length polymorphism analysis of e.g. the urease genes. Furthermore, reproducibility, discriminatory power, ease of performance and interpretation, cost and toxic procedures of each method are assessed. To date no direct comparison of all the molecular typing methods described has been performed in the same study with the same H. pylori strains. However, PCR analysis of the urease gene directly on suspensions of H. pylori or gastric biopsy material seems to be useful for routine use and applicable in specific epidemiological situations.

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.

The growth physiology of Escherichia coli during colonization of the intestinal tract was studied with four animal models: the streptomycin-treated mouse carrying a reduced microflora, the monoassociated mouse with no other microflora than the introduced strain, the conventionalized streptomycin-treated mouse, and the conventionalized monoassociated mouse harboring a full microflora. A 23S rRNA fluorescent oligonucleotide probe was used for hybridization to whole E. coli cells fixed directly after being taken from the animals, and the respective growth rates of E. coli BJ4 in the four animal models were estimated by correlating the cellular concentrations of ribosomes with the growth rate of the strain. The growth rates thus estimated from the ribosomal content of E. coli BJ4 in vivo did not differ in the streptomycin-treated and the monoassociated mice. After conventionalization there was a slight decrease of the bacterial growth rates in both animal models.

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ. In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coil cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coil growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.

Conventional wisdom regarding mechanisms of bacterial pathogenesis holds that pathogens arise by external acquisition of distinct virulence factors, whereas determinants shared by pathogens and commensals are considered to be functionally equivalent and have been ignored as genes that could become adapted specifically for virulence. It is shown here, however, that genetic variation in an originally commensal trait, the FimH lectin of type 1 fimbriae, can change the tropism of Escherichia coli, shifting it toward a urovirulent phenotype. Random point mutations in fimH genes that increase binding of the adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence in the mouse urinary tract. These mutant FimH variants, however, are characterized by increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of E. coli. This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent E. coli. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat.

Salmonella typhimurium SR-11 is extremely virulent at a dose as low as 10(5) colony forming units (cfu) when administered perorally to BALB/c mice. Utilizing mini-transposon mutagenesis, a mutant of S. typhimurium SR-11 was isolated that was unable to utilize oleate and citrate as carbon sources. This mutant, designated S. typhimurium SR-11 Fad- (Fatty acid), was found to utilize sugars under cya/crp control as sole carbon sources, suggesting that the mutation is not in either of these genes. In addition, SR-11 Fad- utilized pyruvate and succinate, but was unable to utilize either acetate or isocitrate as sole carbon source. In contrast to SR-11, SR-11 Fad- was found to be avirulent, i.e. BALB/c mice were completely healthy after oral infection with 10(9) S. typhimurium SR-11 Fad- cells. Moreover, 21 days after SR-11 Fad- infection, BALB/c mice were found to be protected against an oral challenge with 10(9) cells of S. typhimurium SR-11.

Resistance to clarithromycin in H. pylori has emerged in a number of countries. We present the first documented case of H. pylori resistance to clarithromycin in Denmark. This follows an increase in the use of clarithromycin as part of the treatment of H. pylori in the most recent years in Denmark. The need for culturing and susceptibility testing in H. pylori is emphasized.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col- and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae and enhanced E. coli F-18Col- colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX, which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two proteins (22 kDa and 26 kDa) encoded by genes immediately adjacent to leuX.

Escherichia coli F-18, a human fecal isolate, makes type 1 fimbriae in vitro and in the streptomycin-treated mouse large intestine in vivo, and is an excellent colonizer of the cecal mucus layer in the streptomycin-treated mouse large intestine. E. coli F-18(pPKL91) harbors an extra fimB gene on a parB stabilized pPBR322 plasmid and is therefore phase-locked 'on' such that all cells express type 1 fimbriae. E. coli F-18(pPR633) contains essentially the same plasmid minus the fimB gene and in L-broth about 30% of the cells express type 1 fimbriae. When fed alone to streptomycin-treated mice, E. coli F-18(pPKL91) colonized the large intestine at about 10(7) cfu/g of feces. However, when simultaneously fed with E. coli F-18(pPR633) at either high (10(10) cfu), or low doses (10(4) cfu), E. coli F-18(pPKL91) was a poor colonizer dropping to a level of between 10(2) and 10(3) cfu/g of feces. When given enough time to establish the state of colonization (10 days), E. coli F-18(pPKL91) persisted in feces in high numbers despite subsequent challenge by E. coli F-18(pPR633). Moreover, although both E. coli F-18(pPR633) and E. coli F-18(pPKL91) grew equally well in cecal mucus in vitro, E. coli F-18(pPR633) traveled through a layer of cecal mucus in vitro much faster than E. coli F-18(pPKL91). Together, the data suggest that type 1 fimbriated cells are at a disadvantage in initiating the colonization state because they have difficulty entering the mucus layer of the intestine as rapidly as non-fimbriated cells. The data also point to the possible biological significance of type 1 fimbrial phase-variation in the mouse large intestine.

Microbial pathogenicity is a complex phenomenon encompassing many varied mechanisms. There are, however, several common strategies that pathogenic organisms use to sustain themselves and overcome host barriers. In one of these strategies, the first step is the firm adhesion of the microorganism to host cells. Adhesion is frequently mediated by organelles called fimbriae. A surprising variety of fimbrial adhesins have been found in Escherichia coli, and several have been extensively characterized. The genetics and regulation of the various fimbrial structures are reviewed, and strategies for preventing the adhesion of harmful bacteria to host surfaces are discussed.

Three minor components of type 1 fimbriae, FimF, FimG and FimH have been characterized. These proteins are integrated in the fimbrial structure; are responsible for the adhesive properties of the fimbriae but are not necessary for the production of fimbriae. Fimbriae were purified from different clones harbouring various combinations of the fimF, fimG and fimH genes in addition to the fimA gene. The FimF, FimG and FimH proteins were identified by two dimentional gel electrophoresis. They were found to have molecular weights of 18.0 kDa, 17.0 kDa and 30 kDa, respectively. The ratio of FimF, FimG and FimH components to the major subunit was less than 1:100. The fimH protein especially was present in very small quantities. Sera raised against fimbriae from two of the clones (HB101/pPKL5 and HB101/pPKL4) were found by immunoblotting to be specific respectively for the major structural protein only (FimA), and for all components.

Identification of antigenic determinants of K88 fimbriae was approached by immunization of rabbits with BSA conjugated synthetic peptides mimicking either a predicted common determinant or type specific determinants. Anti-peptide sera were assayed by ELISA and sandwich ELISA. It was shown that sera raised against the peptides corresponding to the K88ab and K88ad variants of the 213-219 segment were able to recognize the corresponding antigenic variant of the native fimbriae whereas the rest of the antisera did not to any significant degree react with intact fimbriae. In Western blots all anti-peptide sera were able to recognize the K88 proteins. Similarly in ELISA assays all raised sera showed affinity to denatured K88 proteins.