Molecular analysis of superoxide dismutase in Campylobacter lari

The superoxide dismutase (SOD) gene clusters, sodB and sodC, and their adjacent genetic loci from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain were analyzed molecularly, and compared with those of thermophilic campylobacters. The UPTC CF89-12 strain carried sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) genes, as did the Campylobacter lari RM2100 reference strain. However, the other three thermophilic Campylobacter jejuni, C. coli and C. upsaliensis reference strains carried only a single sodB gene, and no sodC. Although sodB and sodC in the UPTC strain shared relatively high nucleotide sequence similarities (92.9 % and 91.7 %, respectively) with the corresponding genes in the C. lari RM2100 strain, the sodB gene in the UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respectively. All PCR amplifications of sodB and sodC gene segments with 28 C. lari isolates, including 14 UPTC isolates, gave positive results. C. lari organisms were shown to carry both the sodB and sodC genes with extremely high frequency. More high-SOD activity was seen with the C. lari isolates (n?=?9), including UPTC, than was seen with the other three thermophilic Campylobacter and Helicobacter pylori organisms.     

RNase III deficient Salmonella typhimurium LT2 contains intervening sequences (IVSs) in its 23S rRNA

Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90–110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation. In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S. typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12. The resulting strain of S. typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E. coli. These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes.     

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter lari

PCR amplifications using primers for the clustered regularly interspaced short palindromic repeats (CRISPRs)-associated gene 1 (cas1), cas2, putative (p)-cas and CRISPRs genes generated cas1, cas2, p-cas and CRISPRs genes segments with 9–28 of 28 urease-positive thermophilic Campylobacter (UPTC) isolates, respectively. The p-cas and CRISPRs genes segments were amplified with 10 of 11 and 0 of 11 urease-negative (UN) Campylobacter lari isolates, respectively. When the nucleotide sequences of the CRISPRs consensus sequence repeats of each 33–37 base pairs from the 18 Campylobacter jejuni isolates were aligned, as well as from the four C. jejuni reference and UPTC CF89-12 strains, the repeats were identified as being almost identical. Although a total of all 18 C. jejuni isolates examined gave PCR-positive signals for the CRISPRs genes, it was, interestingly, suggested that many numbers of C. lari and C. jejuni isolates may possibly carry cas but not CRISPRs genes within their CRISPRs loci. In addition, PCR amplification by using a novel primer pair of f-ClCRISPR-ladder and ClCRISPRs-R, which were novel to this study, with the UPTC CF89-12 strain was shown to be useful for the detection of the putative CRISPRs separated by the non-repetitive unique spacer regions, with the electrophoretic ladder DNA profile following 5.0 % polyacrylamide gel electrophoresis. Secondary structure models of the CRISPRs repeats were predicted with UPTC CF89-12 and two C. jejuni strains.     

Intervening Sequences in 16S rRNA Genes of Campylobacter sp.: Diversity of Nucleotide Sequences and Uniformity of Location

We found and sequenced intervening sequences (IVSs) in the PCR-amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233 bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9-100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.     

Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia

Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.     

Natural Transformation-Mediated Transfer of Erythromycin Resistance in Campylobacter coli Strains from Turkeys and Swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10⁻⁵ to 10⁻⁶ for turkey-derived strains but 10⁻⁷ or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

In vitro compared activity of telithromycin and azithromycin against northwest Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility

Aims: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005–2007 from specimens of different human origin obtained in three Piemonte Region’s hospitals. Methods and Results: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin‐resistant (Ery‐R) phenotypes by the triple‐disc test. Exactly 78·8% of S. pyogenes and 69·2% of S. pneumoniae were erythromycin‐susceptible (Ery‐S). Concerning S. pyogenes, telithromycin was active against M and inducible MLS_B, subtype‐C, phenotypes but not against constitutive MLS_B strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide‐resistance. On the contrary, the Ery‐R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. Conclusions: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21·2% of S. pyogenes and 30·8% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery‐R streptococcal isolates. Significance and Impact of the Study: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.     

Identification and characterization of intervening sequences within 23S rRNA genes from more than 200 Campylobacter isolates from seven species including atypical campylobacters

Background  

Identification and characterization of intervening sequences (IVSs) within 23S rRNA genes from Campylobacter organisms including atypical campylobacters were carried out using two PCR primer pairs, designed to generate helix 25 and 45 regions.     

Sequence diversity of intervening sequences (IVSs) in the 23S ribosomal RNA in Salmonella spp

Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.     

Gene mutations of 23S rRNA associated with clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients

Although resistance of Helicobacter pylori to clarithromycin is a major cause of failure of eradication therapies, little information is available regarding gene mutations of clarithromycin-resistant primary and secondary H. pylori isolates in Korea. In the present study, we examined gene mutations of H. pylori 23S rRNA responsible for resistance to clarithromycin. DNA sequences of the 23S rRNA gene in 21 primary clarithromycin-resistant and 64 secondary clarithromycin-resistant strains were determined by PCR amplification and nucleotide sequence analyses. Two mutations of the 23S rRNA gene, A2143G and T2182C, were observed in primary clarithromycin-resistant isolates. In secondary isolates, dual mutation of A2143G+T2182C was frequently observed. In addition, A2143G+T2182C+ T2190C, A2143G+T2182C+C2195T, and A2143G+T2182C +A2223G were observed in secondary isolates. Furthermore, macrolide binding was tested on purified ribosomes isolated from T2182C or A2143C mutant strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the mutant strains. These results indicate that secondary isolates show a greater variety of 23S rRNA gene mutation types than primary isolates, and triple mutations of secondary isolates are associated with A2143G+T2182C in H. pylori isolated from Korean patients.     

Identification of shiga toxin-producing bacteria by a new immuno-capture toxin gene PCR

Mechanisms and occurrence of macrolide resistance in the periodontal pathogen Treponema denticola have received little attention. In this study, erythromycin resistance due to mutations in the genes encoding T. denticola 23S rRNA was investigated. The T. denticola genome was shown to contain two copies of 23S rDNA. 23S rRNA genes of nine erythromycin-resistant isolates derived from T. denticola were amplified and sequences were analyzed. All the erythromycin-resistant strains had at least one A-->G transition mutation at the 23S rRNA gene sequence cognate to position A2058 in Escherichia coli 23S rDNA. This suggests that antibiotic pressure is sufficient to select for point mutations that confer resistance in this organism.     

Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.