The Helicobacter pylori genome sequence: genetic factors for long life in the gastric mucosa.
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Animals↗
Biomedical subjects
D E Berg
Publications and source records attributed to D E Berg.
Helicobacter pylori populations in Peruvian patients.
Helicobacter pylori is an extremely diverse species. The characterization of strains isolated from individual patients should give insights into colonization and disease mechanisms and bacterial evolution. We studied H. pylori isolates from patients in the Japanese-Peruvian Polyclinic in Lima, Peru, by determining metronidazole susceptibility or resistance and by random amplified polymorphic DNA (RAPD) fingerprinting (a measure of overall genotype). Strains isolated from several biopsy specimens from each of 24 patients were studied. Both metronidazole-susceptible and -resistant strains were isolated from 13 patients, whereas strains of more than one RAPD type were isolated from only seven patients. We propose that the homogeneity in RAPD fingerprints for strains isolated from most persons reflects selection for particular H. pylori genotypes during chronic infection in individual hosts and the human diversity in traits that are important to this pathogen. Carriage of related metronidazole-resistant and -susceptible strains could reflect frequent metronidazole use in Peru and alternating selection for resistant and susceptible phenotypes during and after metronidazole therapy.
Adult↗
Corrected identity of isolates of Helicobacter pylori reference strain NCTC11637.
BACKGROUND: The reference strains NCTC11637 and NCTC11638 were among the very first Helicobacters ever cultured and have been distributed through national reference culture collections to researchers throughout the world. Because H. pylori is an extremely diverse species, such reference strains are invaluable as universal standards, provided that they are identified correctly. MATERIALS AND METHODS: H. pylori strains (previously called "NCTC11637") from three different sources and NCTC11638 were fingerprinted by the arbitrarily primed polymerase chain reaction (PCR) (also known as random amplified polymorphic DNA, or RAPD) method and further were characterized by NotI digestion and pulsed field gel electrophoresis of total genomic DNA (NotI-PFGE) and by restriction of PCR-amplified ureCD and flaA gene segments. RESULTS: RAPD tests of two "NCTC11637" strains from different sources (CCUG17874, UA1178) indicated that they were closely related or identical to NCTC11638. Given the diversity of H. pylori strains and the high sensitivity of the RAPD method, close matches in RAPD patterns from independent clinical isolates are not expected. In contrast, the version of "NCTC11637" from the American Type Culture Collection (ATCC43504) did not match NCTC11638 in RAPD fingerprint. Concordant results were obtained by NotI-PFGE and by restriction of PCR amplified gene segments. CONCLUSIONS: Two unrelated versions of the reference (type) H. pylori strain NCTC11637 are in general circulation and are distinguished easily by DNA fingerprinting. One matches another reference strain, NCTC11638, whereas the other is distinct from it, as expected of independent clinical isolates. Knowing which "NCTC11637" reference strain one has could be important, especially because H. pylori strains probably are diverse in phenotypic traits that are important for colonization or disease.
Bacterial Typing Techniques↗
Epidemiology of cagA/vacA genes in H. pylori isolated from children and adults in Poland.
Serious lesions in gastric mucosa seem to be a result of infection with more pathogenic H. pylori strains. It was shown that two different proteins: CagA and VacA are the pathogenicity markers of H. pylori. CagA/VacA profile is associated with high intensity of inflammation and development of peptic ulcer disease. Presence of CagA protein depends on possession by a strain of cagA gene. VacA protein is expressed in every strain, but particular alleles of vacA gene are responsible for different levels of cytotoxin production. We estimated frequency of the cagA gene and particular alleles of vacA gene in 84 H. pylori strains by PCR method. Eighty percent of children and 72.4% of adults were infected with cagA(+) H. pylori strains. Presence of cagA gene was correlated with active gastritis in 60% of infected children and adults. Majority of H. pylori strains represented s1m2 and s1m1 vacA alleles' combinations that are responsible for high and medium level of cytotoxin production. Our data may suggest high risk of development of serious consequences of H. pylori infection especially in children treated unsuccessfully.
Adult↗
Genetic diversity of Helicobacter pylori.
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Genetic Variation↗
Transient and persistent experimental infection of nonhuman primates with Helicobacter pylori: implications for human disease.
Helicobacter pylori can establish chronic infection in the human gastric mucosa, and it is a major cause of peptic ulcer disease and a principal risk factor for gastric cancer. This creates a need for H. pylori infection models that mimic the human condition. To test the suitability of rhesus monkeys as infection models, H. pylori-free animals were inoculated intragastrically with mixtures of H. pylori strains, bacteria recovered from colonized animals were typed by arbitrarily primed PCR, and host inflammatory and immunologic responses were monitored. Among five H. pylori-free animals inoculated with a mixture of two human strains plus one monkey strain, one became persistently infected and one became only transiently infected. The recovered bacteria matched the monkey input strain in DNA fingerprint. A subsequent trial using two new human isolates and three animals that had resisted colonization by the monkey strain resulted in persistent infection in one animal and transient infection in two others. Antral gastritis, anti-H. pylori serum immunoglobulin G, and atrophy all increased, but with patterns that differed among animals. We conclude that (i) rhesus monkeys can be infected experimentally with H. pylori, (ii) individuals differ in susceptibility to particular bacterial strains, (iii) infections may be transient, and (iv) the fitness of a particular strain for a given host helps determine the consequences of exposure to that strain.
Animals↗
Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.
Many of the virulence genes of pathogenic strains of Escherichia coli are carried in large multigene chromosomal segments called pathogenicity islands (PAIs) that are absent from normal fecal and laboratory K-12 strains of this bacterium. We are studying PAIs in order to better understand factors that govern virulence and to assess how such DNA segments are gained or lost during evolution. The isolation and sample sequencing of a set of 11 cosmid clones that cover all of one and much of a second large PAI in the uropathogenic E. coli J96 are described. These PAIs were mapped to the 64- and 94-min regions of the E. coli K-12 chromosome, which differ from the locations of three PAIs identified in other pathogenic E. coli strains. Analysis of the junction sequences with E. coli K-12-like DNAs showed that the insert at 94 min is within the 3' end of a phenylalanine tRNA gene, pheR, and is flanked by a 135-bp imperfect direct repeat. Analysis of the one junction recovered from the insert at 64 min indicated that it lies near another tRNA gene, pheV. To identify possible genes unique to these PAIs, 100 independent subclones of the cosmids were made by PstI digestion and ligation into a pBS+ plasmid and used in one-pass sample DNA sequencing from primer binding sites at the cloning site in the vector DNA. Database searches of the J96 PAI-specific sequences identified numerous instances in which the cloned DNAs shared significant sequence similarities to adhesins, toxins, and other virulence determinants of diverse pathogens. Several likely insertion sequence elements (IS100, IS630, and IS911) and conjugative R1 plasmid and P4 phage genes were also found. We propose that such mobile genetic elements may have facilitated the spread of virulence determinants within PAIs among bacteria.
Base Sequence↗
Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.
Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.
Bacterial Proteins↗
Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.
An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.
Animals↗
Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.
We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.
Base Sequence↗
Control of nosocomial infections in an intensive care unit in Guatemala City.
We tested the effectiveness of specific vs. general infection control interventions in a teaching hospital in Guatemala City. After 3 months of prospective surveillance, we implemented targeted interventions (i.e., modification of respiratory tract care and use of a closed urinary catheter drainage system), an educational program focused on respiratory intervention, and general interventions (i.e., aseptic technique). The rate of nosocomial pneumonia, the most common nosocomial infection, decreased from 33% (41 of 123 patients) before intervention to 16% (21 of 130 patients) after intervention (P = .001). Although the frequency of hand washing increased from 5% to 63% (P < .001), the rates of other types of nosocomial infections did not change significantly. The combination of targeted respiratory intervention and an intense, focused educational campaign reduced the rate of nosocomial pneumonia. General improvements in hygiene and hand washing rates, or even implementation of a closed urinary drainage system without focused education, may not be sufficient to reduce infection rates in intensive care units in developing countries.
Adult↗
Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets.
Clinical isolates of Helicobacter pylori, the gastric pathogen implicated in gastritis, peptic ulcers, and gastric cancer in humans, are diverse in traits likely to be important for colonization and disease. Here we report studies using a gnotobiotic piglet-H. pylori infection model to test for host-specific adaptation and to detect cocolonization by different strains. First, an H. pylori strain that initially had grown only weakly in piglets was adapted to them by spontaneous mutation and selection during 12 serial passages; this resulted in an increase in yield from about 10(3) to > 10(7) bacteria per g of mucosa. Second, piglets were fed mixtures of two different well-adapted strains and the presence of one or both strains was monitored by restriction analysis of a PCR-amplified flagellar (flaA) gene segment. The restriction fragment patterns from pools of bacteria indicated that both strains had colonized most piglets and that both strains were present at more than half of the individual biopsy sites, although often at unequal ratios. This suggests a microcolonial mode of growth with limited migration of bacteria between neighboring sites in the gastric mucosa. We propose that the gnotobiotic piglet-H. pylori infection model will be useful for testing how spontaneous mutation, selection, and DNA transfer between strains during mixed infection may each contribute to adaptation to specific hosts and the evolution of virulence of this important pathogen.
Adaptation, Biological↗
Cloning and characterization of hemolytic genes from Helicobacter pylori.
Strains of Helicobacter pylori, the bacterium associated with gastritis, peptic ulcer disease, and gastric cancer in humans, express different degrees of hemolysis on agar containing erythrocytes (RBC). Here we report the isolation and characterization of six recombinant clones from a genomic library of H. pylori ATCC 49503 that confer on Escherichia coli the ability to lyse sheep RBC. DNA hybridizations indicated no sequence homology among these hemolytic clones. Hybridization mapping of them to an ordered H. pylori cosmid library identified their separate chromosomal locations. One clone hybridized to two regions separated by approximately 200 kb. The specificities of the hemolytic activities of these clones were tested with RBC from humans, monkeys, cattle, horses, guinea pigs, rabbits, and chickens as well as with RBC from sheep. One clone conferred the ability to lyse RBC from five species, a second clone allowed the lysis of RBC from four of these species, three other clones allowed the lysis of RBC from three of these species, and the sixth clone allowed the lysis of RBC from just two species. We propose that some or all of the genes that confer these various hemolytic activities contribute to pathogen-host tissue interactions and that the different specificities seen here are important for H. pylori infections of humans of different genotypes or disease states.
Cloning, Molecular↗
Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.
We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth.
Bacterial Proteins↗
Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.
The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin. We conclude that H. pylori genotypes, as measured by several sensitive DNA fingerprinting methods, can remain stable for years in vivo, despite the acquisition or loss of drug resistance, circulating antibody, or exposure to antibiotics or sucralfate.
Anti-Bacterial Agents↗
Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157:H7 strains by using templates from boiled cultures.
Arbitrarily primed PCR allows genetically different bacterial strains to be distinguished with great sensitivity and efficiency. We report that informative, reproducible arbitrarily primed PCR profiles can be obtained from Escherichia coli O157:H7 strains by using boiled stationary-phase cultures, without the need for time-consuming phenol extraction. This simple template preparation procedure should be especially useful in large epidemiologic studies when many strains must be typed.
Bacterial Typing Techniques↗
Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients.
Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency.
Bacterial Typing Techniques↗
Mycoplasma gallisepticum strain differentiation by arbitrary primer PCR (RAPD) fingerprinting.
We demonstrate here that the arbitrary primer polymerase-chain-reaction-based DNA fingerprinting method (also termed random amplified polymorphic DNA or RAPD) can be used to distinguish among strains of the avian pathogen Mycoplasma gallisepticum. Ten base oligonucleotide primers were used individually to prime DNA synthesis from genomic DNAs. Strain-specific arrays of DNA fragments were generated, which allowed us to identify and group isolates. Isolates of M. synoviae, M. gallinarum and M. iners yielded arrays of DNA fragments that differed markedly from those generated from the M. gallisepticum isolates using the same arbitrary primers. These results show that the RAPD fingerprinting method distinguishes genetically different strains of M. gallisepticum and indicates that it should be valuable for monitoring transmission of this pathogen.
Animals↗