Microarray-based genomic surveying of gene polymorphisms in Chlamydia trachomatis

By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis

Nucleotide and phylogenetic analyses of the Chlamydia trachomatis ompA gene indicates it is a hotspot for mutation

<p>Abstract</p> <p>Background</p> <p>Serovars of the human pathogen <it>Chlamydia trachomatis </it>occupy one of three specific tissue niches. Genomic analyses indicate that the serovars have a phylogeny congruent with their pathobiology and have an average substitution rate of less than one nucleotide per kilobase. In contrast, the gene that determines serovar specificity, <it>ompA</it>, has a phylogenetic association that is not congruent with tissue tropism and has a degree of nucleotide variability much higher than other genomic loci. The <it>ompA </it>gene encodes the major surface-exposed antigenic determinant, and the observed nucleotide diversity at the <it>ompA </it>locus is thought to be due to recombination and host immune selection pressure. The possible contribution of a localized increase in mutation rate, however, has not been investigated.</p> <p>Results</p> <p>Nucleotide diversity and phylogenetic relationships of the five constant and four variable domains of the <it>ompA </it>gene, as well as several loci surrounding <it>ompA</it>, were examined for each serovar. The loci flanking the <it>ompA </it>gene demonstrated that nucleotide diversity increased monotonically as <it>ompA </it>is approached and that their gene trees are not congruent with either <it>ompA </it>or tissue tropism. The variable domains of the <it>ompA </it>gene had a very high level of non-synonymous change, which is expected as these regions encode the surface-exposed epitopes and are under positive selection. However, the synonymous changes are clustered in the variable regions compared to the constant domains; if hitchhiking were to account for the increase in synonymous changes, these substitutions should be more evenly distributed across the gene. Recombination also cannot entirely account for this increase as the phylogenetic relationships of the constant and variable domains are congruent with each other.</p> <p>Conclusions</p> <p>The high number of synonymous substitutions observed within the variable domains of <it>ompA </it>appears to be due to an increased mutation rate within this region of the genome, whereas the increase in nucleotide substitution rate and the lack of phylogenetic congruence in the regions flanking <it>ompA </it>are characteristic motifs of gene conversion. Together, the increased mutation rate in the <it>ompA </it>gene, in conjunction with gene conversion and positive selection, results in a high degree of variability that promotes host immune evasion.</p

The ompA Gene in Chlamydia trachomatis Differs in Phylogeny and Rate of Evolution from Other Regions of the Genome

Strains of Chlamydia trachomatis are classified into serovars based on nucleotide sequence differences in ompA, the gene that encodes the major outer membrane protein. Phylogenetic characterization of strains based on ompA, however, results in serovar groupings that are inconsistent with the distinguishing features of C. trachomatis pathobiology, e.g., tissue tropisms and disease presentation. We have compared nucleotide sequences at multiple sites distributed around the chlamydial genome from 18 strains representing 16 serovars; sampled regions included genes encoding housekeeping enzymes (totaling 2,073 bp), intergenic noncoding segments (1,612 bp), and a gene encoding a second outer membrane protein (porB; 1,023 bp), with the ompA sequence (1,194 bp) used for reference. These comparative analyses revealed substantial variation in nucleotide substitution patterns among the sampled regions, with average pairwise sequence differences ranging from 0.15% for the housekeeping genes to 12.1% for ompA. Phylogenetic characterization of the sampled genomic sequences yielded a strongly supported tree that divides the strains into groupings consistent with C. trachomatis biology and which has a topology quite distinct from the ompA tree. This phylogenetic incongruity can be accounted for by recombination of the ompA gene between different genomic backgrounds. We found, however, no evidence of recombination within or between any of the sampled regions around the C. trachomatis genome apart from ompA. Parallel analysis of published sequence data on four members of the pmp gene family are consistent with the phylogenetic analyses reported here

Chloramphenicol and tetracycline decrease motility and increase invasion and attachment gene expression in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium is one of the most common serovars isolated from humans and livestock, and over 35% of these isolates are resistant to three or more antibiotics. Multidrug-resistant (MDR) Salmonella is a public health concern as it is associated with increased morbidity in patients compared to antibiotic sensitive strains, though it is unknown how the antibiotic resistant isolates lead to a more severe infection. Cellular invasion is temporally regulated in Salmonella and normally occurs during late-log and stationary growth. However, our previous work determined that a 30 minute exposure to a sub-inhibitory concentration of tetracycline can induce the full invasion phenotype during early-log growth in certain MDR S. Typhimurium isolates. The current study examined whether sub-inhibitory concentrations of other antibiotics could also induce the invasiveness in the same set of isolates. Ampicillin and streptomycin had no effect on invasion, but certain concentrations of chloramphenicol were found to induce invasion in a subset of isolates. Two of the isolates induced by chloramphenicol were also inducible by tetracycline. RNA-seq analyses demonstrated that chloramphenicol and tetracycline both down-regulated motility gene expression, while up-regulating genes associated with attachment, invasion, and intracellular survival. Eleven fimbrial operons were up-regulated, which is notable as only three fimbrial operons were thought to be inducible in culture; six of these up-regulated operons have been reported to play a role in Salmonella persistence in mice. Overall, these data show that the normal progression of the genetic pathways that regulate invasion can be expedited to occur within 30 minutes due to antibiotic exposure. This altered invasion process due to antibiotics may play a role in the increased intensity and duration of infection observed in patients with MDR Salmonella

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium

Abstract Background Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline and florfenicol are frequently administrated to food-producing animals to treat and prevent various diseases. Therefore, we evaluated the response of MDR S. Typhimurium after exposure to these two antibiotics. Results We exposed four MDR S. Typhimurium isolates to sub-inhibitory concentrations of chlortetracycline (16 and 32 µg/ml) or florfenicol (16 µg/ml) for 30 min during early-log phase. Differentially expressed genes following antibiotic treatment were identified using RNA-seq, and genes associated with attachment and those located within the Salmonella pathogenicity islands were significantly up-regulated following exposure to either antibiotic. The effect of antibiotic exposure on cellular invasion and motility was also assessed. Swimming and swarming motility were decreased due to antibiotic exposure. However, we observed chlortetracycline enhanced cellular invasion in two strains and florfenicol enhanced invasion in a third isolate. Conclusions Chlortetracycline and florfenicol exposure during early-log growth altered the expression of nearly half of the genes in the S. Typhimurium genome, including a large number of genes associated with virulence and pathogenesis; this transcriptional alteration was not due to the SOS response. The results suggest that exposure to either of these two antibiotics may lead to the expression of virulence genes that are typically only transcribed in vivo, as well as only during late-log or stationary phase in vitro

Supershed Escherichia coli O157:H7 Has Potential for Increased Persistence on the Rectoanal Junction Squamous Epithelial Cells and Antibiotic Resistance

Supershedding cattle shed Escherichia coli O157:H7 (O157) at ≥ 104 colony-forming units/g feces. We recently demonstrated that a supershed O157 (SS-O157) strain, SS-17, hyperadheres to the rectoanal junction (RAJ) squamous epithelial (RSE) cells which may contribute to SS-O157 persistence at this site in greater numbers, thereby increasing the fecal O157 load characterizing the supershedding phenomenon. In order to verify if this would be the signature adherence profile of any SS-O157, we tested additional SS-O157 isolates (n = 101; each from a different animal) in the RSE cell adherence assay. Similar to SS-17, all 101 SS-O157 exhibited aggregative adherence on RSE cells, with 56% attaching strongly (>10 bacteria/cell; hyperadherent) and 44% attaching moderately (1–10 bacteria/cells). Strain typing using Polymorphic Amplified Typing Sequences (PATS) analysis assigned the 101 SS-O157 into 5 major clades but not to any predominant genotype. Interestingly, 69% of SS-O157 isolates were identical to human O157 outbreak strains based on pulsed field gel electrophoresis profiles (CDC PulseNet Database), grouped into two clades by PATS distinguishing them from remaining SS-O157, and were hyperadherent on RSE cells. A subset of SS-O157 isolates (n = 53) representing different PATS and RSE cell adherence profiles were analyzed for antibiotic resistance (AR). Several SS-O157 (30/53) showed resistance to sulfisoxazole, and one isolate was resistant to both sulfisoxazole and tetracycline. Minimum inhibitory concentration (MIC) tests confirmed some of the resistance observed using the Kirby–Bauer disk diffusion test. Each SS-O157 isolate carried at least 10 genes associated with AR. However, genes directly associated with AR were rarely amplified: aac (3)-IV in 2 isolates, sul2 in 3 isolates, and tetB in one isolate. The integrase gene, int, linked with integron-based AR acquisition/transmission, was identified in 92% of SS-O157 isolates. Our results indicate that SS-O157 isolates could potentially persist longer at the bovine RAJ but exhibit limited resistance towards clinical antibiotics