High frequency of co-resistance in CTX-M-producing Escherichia coli to non-beta-lactam antibiotics, with the exceptions of amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin, in a county of Sweden.

BACKGROUND: The objective of this study was to investigate the in vitro activity of different antibiotics against CTX-M-producing Escherichia coli in a county of Sweden, and to determine the occurrence of multi-resistance and plasmid- mediated quinolone resistance among these isolates. METHODS: A total of 198 isolates of E. coli with extended-spectrum beta-lactamase (ESBL) phenotype and mainly CTX-M genotype were studied. The minimum inhibitory concentrations (MICs) for amikacin, chloramphenicol, ciprofloxacin, colistin, fosfomycin, gentamicin, nalidixic acid, nitrofurantoin, tigecycline, tobramycin, trimethoprim, and trimethoprim-sulfamethoxazole were determined with the Etest. Susceptibility was defined according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MIC50 and MIC90 values were calculated. RESULTS: Ninety-five percent or more of the isolates were susceptible to amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin. CTX-M group 9 was more susceptible than CTX-M group 1 to ciprofloxacin, gentamicin, and tobramycin. Sixty-eight percent of the isolates were multi-resistant, and the most common multi-resistance pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and tobramycin. Only 1 isolate carried a qnrS1 gene, but 37% carried aac(6')-Ib-cr. CONCLUSIONS: A high frequency of co-resistance between ESBL-producing E. coli and non-beta-lactam antibiotics was seen. On the other hand, very high susceptibility was seen for amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin. These data support the replacement of gentamicin and tobramycin, normally used in Sweden, with amikacin, for severe infections.

Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden.

BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes. RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed. CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae and trends in antibiotic consumption in a county of Sweden.

In the last decade extended-spectrum beta-lactamase (ESBL)-producing bacteria have become an increasing problem. Our aims were to investigate the prevalence of ESBL-producing Enterobacteriaceae and trends in antibiotic use in the county of Östergötland, Sweden. From 2002 through 2007 there were 224 ESBL-producing Escherichia coli and 23 Klebsiella pneumoniae isolates with an ESBL-phenotype identified among all Enterobacteriaceae isolated at the clinical laboratory. Trends in antibiotic consumption expressed as defined daily doses (DDD) per 1000 inhabitants and day (DID) were studied. The prevalence of ESBL-producing isolates among Enterobacteriaceae in our region is still low (< 1%). Patients with ESBL-producing E. coli increased significantly (p < 0.001) from 5 in y 2002 to 47 in y 2007. CTX-M group 1 was the dominant enzyme group in both E. coli and K. pneumoniae. Antibiotic susceptibility testing of ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole revealed that 58% of E. coli and 50% of K. pneumoniae isolates were multi-resistant. Antibiotic use remained unchanged from 2001 through 2009, but there was a trend towards increased use of drugs with low ESBL selection potential, which was probably due to the increased prevalence of ESBL producers.

Molecular detection of aggregation substance, enterococcal surface protein, and cytolysin genes and in vitro adhesion to urinary catheters of Enterococcus faecalis and E. faecium of clinical origin.

It has been hypothesized that nosocomial enterococci might have virulence factors that enhance their ability to colonise hospitalised patients. The objectives of this study were to investigate the prevalence of genes encoding 3 virulence factors: aggregation substance (asa1), enterococcal surface protein (esp), and 5 genes within the cytolysin operon (cylA, cylB, cylM, cylL(L), cylL(S)) and cytolysin production in 115 enterococcal clinical isolates (21 Enterococcus faecium and 94 E. faecalis). Adhesion to siliconized latex urinary catheters in relation to presence of esp was analysed in a subset of isolates. The isolates were previously characterised by pulsed-field gel electrophoresis (PFGE). esp was the only virulence gene found in E. faecium. It was found in 71% of the 21 E. faecium isolates. asa1, esp, and the cyl operon were found in 79%, 73% and 13% respectively, of the 94 E. faecalis isolates. There was a complete agreement between presence of the cyl operon and phenotypic cytolysin production. Isolates belonging to a cluster of genetically related isolates carried esp and asa1 more often when compared to unique isolates. No difference was found with respect to cyl genes. E. faecalis isolates adhered with higher bacterial densities than E. faecium. E. faecalis isolates within the same PFGE cluster adhered with similar bacterial densities, but there was no association between adhesion and the presence of esp when isolates within the same cluster were compared. In conclusion, E. faecalis isolates with high-level gentamicin resistance (HLGR) belonging to clusters of genetically related isolates widely distributed in Swedish hospitals, were likely to carry both esp and asa1. Adhesion was not affected by esp.

Molecular identification of (bla)SHV, (bla)LEN and (bla)OKP beta-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged (bla)SHV-PCR amplicons.

Plasmid encoded (bla)SHV enzymes represent an important sub-group of class A beta-lactamases causing an ESBL-phenotype which is increasingly found in Enterobacteriaceae including Klebsiella pneumoniae. The chromosomally encoded beta-lactamase (bla)LEN and (bla)OKP enzymes, which so far only have been found in K. pneumoniae, do not hydrolyse extended-spectrum cephalosporins. In the present study, multiple displacement amplified DNA derived from 20 K. pneumoniae clinical isolates with a (bla)SHV-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of (bla)SHV, (bla)LEN and (bla)OKP genes was obtained by bi-directional amplicon sequencing using SP6- and T7-specific primers. Three well characterised K. pneumoniae strains having a SHV-genotype were included in the study. The bi-directional amplicon sequencing, covering approximately 800 bp (approximately 93%) of the (bla)SHV, (bla)LEN and (bla)OKP enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of (bla)SHV allelic variants in six K. pneumoniae isolates in which the amplicons had to be cloned accordingly. Based on deduced amino-acid sequences, a dendrogram was constructed. Seventeen out of 20 K. pneumoniae isolates with an ESBL-phenotype formed a SHV-like cluster, two were LEN-like, and one isolate was OKP-like. The PCR-based molecular typing method described here enables a rapid, reliable and cost-effective identification and differentiation of (bla)SHV, (bla)OKP and (bla)LEN genes.

Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.

BACKGROUND: Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A beta-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment. METHODS: Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer. RESULTS: Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and blaOXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae. CONCLUSION: The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.

Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA.

BACKGROUND: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. RESULTS: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. CONCLUSION: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.

Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: identification of cytokine polymorphism by pyrosequencing analysis.

BACKGROUND: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. METHODS: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. RESULTS: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B -511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B -31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. CONCLUSIONS: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.

Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract.

AIM: To investigate the expression of gastrin-releasing peptide (GRP) and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. METHODS: Poly A(+) mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA (ss-cDNA). PCR amplifications were carried out using gene-specific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. RESULTS: Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, co-expression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. CONCLUSION: GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.

Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis.

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis.

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.

Molecular cloning of a putative Ciona intestinalis cionin receptor, a new member of the CCK/gastrin receptor family.

Cionin, a peptide showing similarities with cholecystokinin and gastrin has been shown to be expressed in the gut and neural ganglion of the protochordate Ciona intestinalis. The present report describes the cloning of a putative cionin receptor (CioR), a new member of the CCK/gastrin family from the gastrointestinal tract of C. intestinalis. mRNA from the stomach of C. intestinalis was isolated using a modified RNA extraction procedure and, subsequently, reverse-transcribed into single-stranded cDNA by means of rapid amplification of 5'- and 3'-cDNA ends (RACE-PCR), followed by full-length PCR amplification. The cloned full-length PCR amplicons contained a short upstream open-reading frame (uORF) coding for a putative 16 amino acid long peptide, followed by a long open reading frame encoding a 526 amino acid putative CioR protein. At the amino acid level, the putative CioR protein shared 35-40% homology with cloned mammalian, chicken, and Xenopus laevis CCK receptors. Phylogenetic analysis revealed that the chicken and X. laevis CCK receptors are orthologues of the mammalian CCK2 receptors whereas CioR protein forms a clade with vertebrate cholecystokinin receptors. Moreover, we found that the CioR cDNA and deduced amino acid sequences were found to correspond to the annotated CCK/gastrin-like receptor gene on Scaffold 117 (C. intestinalis draft genome project, Joint Genome Institute database; http://www.jgi.doe.gov).

Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments.

The rapid identification of the etiological agent of microbial infections can bring about both clinical and financial benefits. Thus, fast and generally applicable classification methods are needed that will enable us to rapidly distinguish pathogenic bacteria from commensals or saprophytic bacteria found in the same habitat. We here show that provisional classification of bacterial isolates can be performed on a large scale based on 16S rRNA sequence comparisons using Pyrosequencing, a recently described real-time DNA sequence analysis technique, and the concept of signature matching. The probes we have developed, together with the new technology, will enable early diagnosis of specific pathogens, which is critical for the rational use of antimicrobial therapy in clinical medicine.

Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons.

Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, we have found contaminating bacterial DNA in our reagents used for PCR. Identified bacteria are the water-borne bacterial genera Pseudomonas, Stenotrophomonas, Xanthomonas, Ralstonia and Bacillus. Our results are in concordance with recent reports of contaminated industrial water systems. In light of this conclusion, we believe that there is a need for increased awareness of possible contamination in uncertified widely used molecular biology reagents, including ultra-pure water. Since sequence-based 16S rDNA techniques are used in a variety of settings for bacterial typing and the characterization of microbial communities, we feel that future certification of molecular biology reagents, as free of nucleic acids, would be advantageous.

Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study.

This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared approximately 50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, L-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: a polymerase chain reaction study.

BACKGROUND/AIM: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. MATERIAL AND METHODS: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malmö University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70 degrees C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A(+) mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. RESULTS: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. CONCLUSION: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown.

Pharmacological analysis of CCK(2) receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK(2) receptor.

A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.

Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR.

The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis.

The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.