Barbora Štaudová

Whole genome sequence of the Treponema pallidum subsp. endemicum strain Bosnia A: The genome is related to yaws treponemes but contains few loci similar to syphilis treponemes

Background T. pallidum subsp. endemicum (TEN) is the causative agent of bejel (also known as endemic syphilis). Clinical symptoms of syphilis and bejel are overlapping and the epidemiological context is important for correct diagnosis of both diseases. In contrast to syphilis, caused by T. pallidum subsp. pallidum (TPA), TEN infections are usually spread by direct contact or contaminated utensils rather than by sexual contact. Bejel is most often seen in western Africa and in the Middle East. The strain Bosnia A was isolated in 1950 in Bosnia, southern Europe. Methodology/Principal Findings The complete genome of the Bosnia A strain was amplified and sequenced using the pooled segment genome sequencing (PSGS) method and a combination of three next-generation sequencing techniques (SOLiD, Roche 454, and Illumina). Using this approach, a total combined average genome coverage of 513× was achieved. The size of the Bosnia A genome was found to be 1,137,653 bp, i.e. 1.6–2.8 kbp shorter than any previously published genomes of uncultivable pathogenic treponemes. Conserved gene synteny was found in the Bosnia A genome compared to other sequenced syphilis and yaws treponemes. The TEN Bosnia A genome was distinct but very similar to the genome of yaws-causing T. pallidum subsp. pertenue (TPE) strains. Interestingly, the TEN Bosnia A genome was found to contain several sequences, which so far, have been uniquely identified only in syphilis treponemes. Conclusions/Significance The genome of TEN Bosnia A contains several sequences thought to be unique to TPA strains; these sequences very likely represent remnants of recombination events during the evolution of TEN treponemes. This finding emphasizes a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.

Bacteriocin-encoding genes and ExPEC virulence determinants are associated in human fecal Escherichia coli strains

BackgroundA set of 1181 E. coli strains of human fecal origin isolated in the South Moravia region of the Czech Republic was collected during the years 2007–2010. Altogether, 17 virulence determinants and 31 bacteriocin-encoding genes were tested in each of them.ResultsThe occurrence of bacteriocin-encoding genes was found to be positively correlated with the occurrence of E. coli virulence factors. Based on the presence of virulence factors and their combinations, E. coli strains were classified as non-pathogenic E. coli (n = 399), diarrhea-associated E. coli (n = 179) and ExPEC strains (n = 603). Non-pathogenic and diarrhea-associated E. coli strains had a low frequency of bacteriocinogeny (32.6% and 36.9%, respectively). ExPEC strains encoding S-fimbriae (sfa), P-fimbriae (pap) and having genes for aerobactin biosynthesis (aer, iucC), α-hemolysis (α-hly) and cytotoxic necrosis factor (cnf1) were often bacteriocinogenic (73.8%), had a high prevalence of bacteriocin multi-producers and showed a higher frequency of genes encoding microcins H47, M, V, B17 and colicins E1, Ia and S4.ConclusionsThe occurrence of bacteriocin-encoding genes and ExPEC virulence determinants correlate positively in E. coli strains of human fecal origin. Bacteriocin synthesis appears to modulate the ability of E. coli strains to reside in the human intestine and/or the virulence of the corresponding strains.

Microcin determinants are associated with B2 phylogroup of human fecal Escherichia coli isolates

Escherichia coli strains are classified into four main phylogenetic groups (A, B1, B2, and D) and strains of these phylogroups differ in a number of characteristics. This study tested whether human fecal E. coli isolates belonging to different phylogroups differ in prevalence of bacteriocinogenic isolates and prevalence of individual bacteriocinogenic determinants. A set of 1283 fecal E. coli isolates from patients with different diseases was tested for the presence of DNA regions allowing classification into E. coli phylogroups and for the ability to produce bacteriocins (23 colicins and 7 microcins). Of the isolates tested, the most common was phylogroup B2 (38.3%) followed by phylogroups A (28.3%), D (26.3%) and B1 (7.2%). Altogether, 695 bacteriocin producers were identified representing 54.2% of all tested isolates. The highest prevalence of bacteriocin producers was found in group B2 (60.3%) and the lowest in group B1 (44.6%). Determinants encoding colicins E1, Ia, and microcin mV were most common in phylogroup A, determinants encoding microcins mM and mH47 were most common in phylogroup B2, and determinant encoding mB17 was most common in phylogroup D. The highest prevalence of bacteriocinogeny was found in phylogroup B2, suggesting that bacteriocinogeny and especially the synthesis of microcins was associated with virulent and resident E. coli strains.

P1.011 Whole Genome Sequence of the Treponema Pallidum Ssp. Endemicum, Strain Bosnia A

Background Treponema pallidum ssp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). The TEN Bosnia A strain was isolated in 1950 from a patient’s penile lesion in northeastern Bosnia. Methods To define genetic differences between TEN Bosnia A and other pathogenic treponemes including the agents of syphilis ( T. pallidum ssp. pallidum, TPA)and yaws ( T. pallidum ssp. pertenue, TPE), a high quality sequence of the Bosnia A genome was determined using 454-pyrosequencing, Illumina, SOLiD and traditional Sanger sequencing. Combined average coverage of these sequencing methods was greater than 340x. Results Compared to other TPA and TPE treponemes, the genome of Bosnia A (1,137,653 bp) was smaller in size (∼2 kb) but structurally almost identical to other TPA and TPE strains. The Bosnia A genome clustered with TPE strains (nucleotide identity excluding indels ranged between 99.91 – 99.94%) while TPAstrains were more distantly related (99.79 – 99.82%). More than 400 Bosnia A-specific nt changes (i.e. sequences different from TPA and TEN genomes) were found as the result of our analysis. Conclusions The Bosnia A genome showed similar genetic characteristics as other TPA and TPE strains. Genetic differences found between TPA strains and Bosnia A genome could be used for identification of potential virulence factors of syphis treponemes. Moreover, genetic changes specific for Bosnia A genome could help develop molecular diagnostic tests for endemic syphilis.

P1.009 Analysis of Simple Sequence Repeats in the Genomes of Pathogenic Treponemal Strains

Background Simple sequence repeats (SSR) are repetitive sequences of 1–6 base pairs in DNA which account for genotypic switching (phenotypic variation) through mechanism of polymerase slippage. This mechanism is common in pathogenic bacteria with reduced genome. Methods Msatfinder v2.0.9 was used to identify SSR sequences in the genomes of five strains of Treponema pallidum subsp. pallidum (agent of syphilis); three strains of T. p. subsp. pertenue (agent of yaws); the Bosnia A strain of T. p. subsp. endemicum(agent of endemic syphilis); the Cuniculi A strain of T. paraluiscuniculi (agent of rabbit syphilis); and the Fribourg-Blanc strain (simian isolate genetically close to agent of yaws). Analysis of Solexa data and subsequent cloning approach were used for determination of intrastrain variability. Results Sequence data analysis of 11 treponemal genomes revealed high degree of variability in guanosine/cytosine homopolymeric tracts (G/C-regions) among pathogenic treponemal strains. Altogether, 120 G/C-regions (containing > 7 nucleotides) were found, from which 53 regions showed no variability, 26 represented nucleotide substitution differences, and 41 contained variable numbers of G/Cs. From these 41 regions, 25 were located in intergenic regions and 16 affected ORFs. Interestingly, variable regions were located upstream or within genes coding for Tpr proteins, potential virulence factors, and hypothetical proteins. Furthermore, 30 regions were specific for Cuniculi A strain, 5 regions differentiated pallidum strains from other strains, and 3 regions were specific for pertenue strains. Moreover, additional variability within treponemal strains was found in 54 G/C-regions. Conclusions Inter- and intrastrain variability of G/C-regions may play a significant role in pathogenesis of treponemal diseases. Further experimental studies are needed to verify this hypothesis.