Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Restrição do 16S-23S DNAr intergênico para avaliação da diversidade de Azospirillum amazonense isolado de Brachiaria spp.

O objetivo deste trabalho foi avaliar a diversidade intra-específica de isolados de Azospirillum amazonense e estabelecer a possível influência de diferentes espécies de Brachiaria ssp. e diferentes condições edafoclimáticas. A caracterização da diversidade desses isolados foi conduzida, utilizando-se a análise de restrição da região intergênica 16S-23S DNAr. As estirpes estudadas separaram-se em dois grupos, definidos a 56% de similaridade. As espécies de Brachiaria ssp. influenciaram a diversidade de estirpes. A maioria dos isolados oriundos de B. decumbens e B. brizantha está inserida no primeiro grupo, enquanto os oriundos de B. humidicola concentram-se no segundo grupo.

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(LyS(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Val(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC)) -tRNA(Val(UAC)) -tRNA(Ala(UGC)) and tRNA(Glu(UUC)) -tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and inter-cistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), R damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).

Identification of Staphylococcus saprophyticus isolated from patients with urinary tract infection using a simple set of biochemical tests correlating with 16S-23S interspace region molecular weight patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Análise filogenética de linhagens de Xylella fastidiosa de diferentes hospedeiros baseada na sequencia da região intergênica 16S-23S rDNA

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Filogenia molecular de cianobactérias baseada em sequências do 16S-23S-ITS rDNA e PC-IGS: investigação de transferência lateral do PC

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

PHYLOGENETIC STUDY OF GEITLERINEMA AND MICROCYSTIS ( CYANOBACTERIA) USING PC-IGS AND 16S-23S ITS AS MARKERS: INVESTIGATION OF HORIZONTAL GENE TRANSFER

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análisis de la región intergénica ARNr 16-23S y del gen ARNr 23S en cepas de campo de Mycroplasma hyopneumoniae : estudio filogenético de los micoplasmas porcinos en base al gen ARNr 23S

Programa de doctorado: Sanidad animal

Clinical and microbiological aspects of linezolid resistance mediated by the cfr gene encoding a 23S rRNA methyltransferase.

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^