RESUMEN
The performance of a commercially available Seegene Seeplex STI Master Panel 3 multiplex PCR for Candida species identification was compared with an internal transcribed spacer 2 (ITS2) PCR assay. We found that the Seeplex assay was specific for identification of C. albicans, C. krusei, C. parapsilosis, C. glabrata, C. tropicalis and C. dubliniensis.
Asunto(s)
Candida/clasificación , Candida/aislamiento & purificación , Candidiasis/microbiología , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Técnicas Microbiológicas/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Candida/genética , ADN de Hongos/química , ADN Espaciador Ribosómico/química , Electroforesis Capilar/métodos , Humanos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Escherichia coli/efectos de los fármacos , beta-Lactamasas/biosíntesis , Farmacorresistencia Bacteriana , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/epidemiología , Humanos , Pruebas de Sensibilidad Microbiana , Estadísticas no Paramétricas , Suecia/epidemiologíaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Biopsia , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Genotipo , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Suecia , Factores de Virulencia/genéticaRESUMEN
The mosaic structure of the cagA gene has been suggested to affect Helicobacter pylori CagA-associated pathogenesis. An improved polymerase chain reaction assay allowed for a rapid and detailed molecular analysis of the cagA gene 3'-region in a single amplification step, followed by amplicon sequencing using universal M13 and T7 sequencing primers.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Cartilla de ADN/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Análisis de Secuencia de ADNRESUMEN
The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Secuencias de Aminoácidos/genética , Técnicas de Tipificación Bacteriana , Biopsia , Clonación Molecular , ADN Bacteriano/genética , Genotipo , Helicobacter pylori/patogenicidad , Humanos , Fosforilación , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Manejo de Especímenes , VirulenciaRESUMEN
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.
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Brotes de Enfermedades , Infecciones por Escherichia coli/epidemiología , Escherichia coli/clasificación , Escherichia coli/genética , Tipificación Molecular/métodos , beta-Lactamasas/metabolismo , Anciano , Análisis por Conglomerados , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Genotipo , Humanos , Epidemiología Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Temperatura de TransiciónRESUMEN
BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.
RESUMEN
Molecular typing of Klebsiella species has become important for monitoring dissemination of ß-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Klebsiella/clasificación , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Dermatoglifia del ADN/métodos , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Genotipo , Humanos , Klebsiella/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
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.
Asunto(s)
Antibacterianos/uso terapéutico , Utilización de Medicamentos/estadística & datos numéricos , Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/efectos de los fármacos , beta-Lactamasas/biosíntesis , beta-Lactamas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/farmacología , Niño , Preescolar , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Prevalencia , Suecia/epidemiología , Adulto Joven , beta-Lactamas/farmacologíaRESUMEN
BACKGROUND: The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. FINDINGS: MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. CONCLUSION: Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.
RESUMEN
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.
Asunto(s)
Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Reacción en Cadena de la Polimerasa/métodos , beta-Lactamasas/genética , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana/genética , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Klebsiella oxytoca/enzimología , Klebsiella oxytoca/genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Reacción en Cadena de la Polimerasa/métodos , beta-Lactamasas/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella oxytoca/aislamiento & purificación , Klebsiella pneumoniae/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Plásmidos , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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.
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Adhesión Bacteriana , Proteínas Bacterianas/genética , Cateterismo , Enterococcus faecalis/fisiología , Enterococcus faecium/fisiología , Microbiología Ambiental , Factores de Virulencia/genética , Análisis por Conglomerados , Dermatoglifia del ADN , Electroforesis en Gel de Campo Pulsado , Enterococcus faecalis/clasificación , Enterococcus faecalis/genética , Enterococcus faecalis/aislamiento & purificación , Enterococcus faecium/clasificación , Enterococcus faecium/genética , Enterococcus faecium/aislamiento & purificación , Genotipo , Hospitales , Humanos , SueciaRESUMEN
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.
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Citocinas/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Polimorfismo de Nucleótido Simple , Factores de Virulencia/genética , Adulto , Anciano , Técnicas de Tipificación Bacteriana/métodos , Biopsia , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Gastritis/genética , Gastritis/microbiología , Gastritis/patología , Genes Bacterianos , Genes de ARNr , Genoma Bacteriano , Genoma Humano , Genotipo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/clasificación , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Estómago/microbiología , Estómago/patología , Virulencia/genéticaAsunto(s)
Bacterias/clasificación , Bacterias/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/historia , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Historia del Siglo XXI , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
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.
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Citocinas/sangre , Citocinas/genética , ADN/genética , ADN/aislamiento & purificación , Polimorfismo Genético/genética , Secuencia de Bases , Biopsia , Donantes de Sangre , ADN/sangre , Helicobacter pylori/genética , Humanos , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
BACKGROUND: It has been shown that the non-opioid effects of Met-enkephalin, which is derived from proenkephalin-A, are mediated through a specific opioid growth factor (OGF) receptor which is assumed to be involved in the control of cell growth. The expression and tissue location of proenkephalin-A mRNA in the gastrointestinal tract remains largely unknown. METHODS: In this study we have analyzed the expression of proenkephalin-A mRNA in the human esophagus, gastrointestinal tract and surrounding organs by means of reverse-transcriptase PCR (RT-PCR). RESULTS: The present study demonstrates proenkephalin-A mRNA expression in the human esophagus, gastrointestinal tract, pancreas, and gallbladder. CONCLUSION: The present study demonstrates proenkephalin-A mRNA expression in regions of the human esophagus, gastrointestinal tract, pancreas, and gallbladder tissues which provides information for the future mapping of proenkephalin-A mRNA and protein expression/co-expression at the cellular level.
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Encefalinas/metabolismo , Tracto Gastrointestinal/metabolismo , Precursores de Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Vesícula Biliar/metabolismo , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Páncreas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Péptido Liberador de Gastrina/genética , Tracto Gastrointestinal/metabolismo , ARN Mensajero/análisis , Receptores de Bombesina/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. MATERIALS AND METHODS: DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. RESULTS: Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. CONCLUSIONS: We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Cálculos Biliares/microbiología , Helicobacter/clasificación , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Adulto , Anciano , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Ribosómico , Femenino , Helicobacter/genética , Helicobacter/aislamiento & purificación , Infecciones por Helicobacter/microbiología , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodosRESUMEN
OBJECTIVE: Epidemiological studies have shown that Helicobacter pylori infection with associated chronic gastritis is the main risk factor for development of gastric cancer. The aim of this study was to investigate the long-term development of H. pylori-induced gastritis in Mongolian gerbils in terms of morphology, gastrin secretion, epithelial proliferation and gene expression of pro-inflammatory cytokines. MATERIAL AND METHODS: A total of 133 gerbils were inoculated with H. pylori and 62 served as controls. The gerbils were killed at different time-points between 6 and 94 weeks after inoculation. Serum concentrations of anti-H. pylori IgG and gastrin were determined by enzyme-linked immunoabsorbent assay (ELISA) and radioimmunoassay (RIA), respectively. Epithelial proliferation was evaluated immunohistochemically after labeling with 5-bromo-2'-deoxy-uridine. Gene expression of beta-actin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Histological parameters of gastritis were assessed semiquantitatively and expressed as a "gastritis score". RESULTS: Serum concentrations of anti-H. pylori IgG and gastrin increased over time. Epithelial proliferation in the antrum was increased 6 weeks after inoculation, followed by increased proliferation in the corpus 32 weeks after inoculation. Gene expression of IL-1beta and TNF-alpha were increased in H. pylori-infected gerbils. Beta-actin was not a reliable endogenous control for RT-PCR. With time, gastritis expanded from the antrum to the corpus and the gastritis score increased to reach a peak 32 weeks after inoculation. Pseudopyloric metaplasia (loss of specialized cells) was a characteristic feature in the corpus mucosa. Gastric ulcers, but neither dysplasia nor carcinoma, were observed during 94 weeks of infection. CONCLUSIONS: Long-term H. pylori infection in Mongolian gerbils led to progressive gastritis, glandular atrophy, hypergastrinemia, increased epithelial proliferation and elevated gene expression of pro-inflammatory cytokines.