A 2-pyridone amide inhibitor of transcriptional activity in Chlamydia trachomatis.

Chlamydia trachomatis is a strict intracellular bacterium that causes sexually transmitted infections and eye infections that can lead to life-long sequelae. Treatment options are limited to broad-spectrum antibiotics that disturb the commensal flora and contribute to selection of antibiotic-resistant bacteria. Hence, development of novel drugs that specifically target C. trachomatis would be beneficial. 2-pyridone amides are potent and specific inhibitors of Chlamydia infectivity. The first generation compound KSK120, inhibits the developmental cycle of Chlamydia resulting in reduced infectivity of progeny bacteria. Here, we show that the improved, highly potent second-generation 2-pyridone amide KSK213 allowed normal growth and development of C. trachomatis and the effect was only observable upon re-infection of new cells. Progeny elementary bodies (EBs) produced in the presence of KSK213 were unable to activate transcription of essential genes in early development and did not differentiate into the replicative form, the reticulate body (RB). The effect was specific to C. trachomatis since KSK213 was inactive in the closely related animal pathogen C. muridarum and in C. caviae The molecular target of KSK213 may thus be different in C. trachomatis or non-essential in C. muridarum and C. caviae Resistance to KSK213 was mediated by a combination of amino acid substitutions in both DEAD/DEAH RNA helicase and RNAse III, which may indicate inhibition of the transcriptional machinery as the mode of action. 2-pyridone amides provide a novel antibacterial strategy and starting points for development of highly specific drugs for C. trachomatis infections.

Polymorphisms in MC1R and ASIP Genes are Associated with Coat Color Variation in the Arabian Camel.

Pigmentation in mammals is primarily determined by the distribution of eumelanin and pheomelanin, the ratio of which is mostly controlled by the activity of melanocortin 1 receptor (MC1R) and agouti signaling protein (ASIP) genes. Using 91 animals from 10 Arabian camel populations, that included the 4 predominant coat color phenotypes observed in the dromedary (light brown, dark brown, black, and white), we investigated the effects of the MC1R and ASIP sequence variants and identified candidate polymorphisms associated with coat color variation. In particular, we identified a single nucleotide polymorphism (SNP), found in the coding region of MC1R (901C/T), linked to the white coat color, whereas a 1-bp deletion (23delT/T) and a SNP (25G/A) in exon 2 of ASIP are associated with both black and dark-brown coat colors. Our results also indicate support that the light-brown coat color is likely the ancestral coat color for the dromedary. These sequence variations at the MC1R and ASIP genes represent the first documented evidence of candidate polymorphisms associated with Mendelian traits in the dromedary.

Complete genome sequence of Borrelia crocidurae.

We announce the draft genome sequence of Borrelia crocidurae (strain Achema). The 1,557,560-bp genome (27% GC content) comprises one 919,477-bp linear chromosome and 638,083-bp plasmids that together carry 1,472 open reading frames, 32 tRNAs, and three complete rRNAs, with almost complete colinearity between B. crocidurae and Borrelia duttonii chromosomes.

Sex Differences in Fecal Microbiome Composition and Function of Dromedary Camels in Saudi Arabia.

The gastrointestinal microbiome plays a significant role in diet digestion and the energy production of its host. Several factors that affect the gastrointestinal microbiota composition were studied in camels. Yet, the impact of sex on the gastrointestinal bacteriome of camels remains unexplored to date. In this perspective, the fecal microbiome community composition from dromedary camels was determined in 10 male and 10 female samples using the 16S rRNA amplicon, in order to estimate if this was influenced by sex. The core microbiome in females contained 284 bacterial OTUs and one archaeal OUT, whereas in males, it contained 279 bacterial OTUs and one archaeal OTU. In females, Bacteroidetes and Spirochaetes were significantly more abundant than in male camels, whereas Lentisphaerae and Euryarchaeota were significantly abundant in males. According to Principal Coordinate Analysis and UPGMA clustering, grouping with respect to sex was observed. The functional prediction results showed differences such as energy production and conversion, and that the cell wall/membrane/envelope were enriched in female camels. The fecal microbiome of male camels was rich in amino acid, lipid transport and metabolism.

Genomic differences among strains of Corynebacterium cystitidis isolated from uterus of camels.

INTRODUCTION: Members of the Corynebacterium cystitidis species are usually isolated from kidney and urine of cow having pyelonephritis. Nevertheless, we have isolated Corynebacterium cystitidis for the first time from uterus of camels, extending the type of mammalian host for this species. Furthermore, it remains unknown whether there are significant genetic variations between strains isolated from different host species and anatomic sites. In this perspective, we investigated the genomic diversity of Corynebacterium cystitidis species, whose pan genome remain unexplored to date. METHODOLOGY: Thus, we sequenced and compared the genomes of five Corynebacterium cystitidis of camel origin and a public genome of cow associated Corynebacterium cystitidis. RESULTS: Results revealed open pan genome of 4,038 gene clusters and horizontal gene transfer played a role in the extensive genetic diversity. Further, we found an obvious distinction between cow and camel associated C. cystitidis via phylogenomic analysis and by average nucleotide identity value of 95% between the two distant lineages and > 99% within camel associated C. cystitidis strains. Moreover, our data supports the hypothesis that the gene repertoire of cow associated Corynebacterium cystitidis developed so as to become more adaptable to the urine milieu. These genetic potentials are specifically evident for genes required for benzoate breakdown, iron transport, citrate and alanine utilization. CONCLUSIONS: Our findings confirm the differentiation of strains into camel lineage and cow lineage. These different niches, comprising the uterus of camel and urinary tract of cow probably played a role in shaping the gene repertoire of strains.

Low genetic diversity among Francisella-like endosymbionts within different genotypes of Hyalomma dromedarii ticks infesting camels in Saudi Arabia.

BACKGROUND AND AIM: Hyalomma dromedarii ticks are vectors of disease agents and hosts of Francisella-like endosymbionts (FLEs). Knowledge about intraspecific genetic variation among H. dromedarii and its Francisella species is limited. The aims of this study were to investigate whether certain H. dromedarii genotypes are specialized in carrying specific Francisella species genotypes and scrutinize the population structure of H. dromedarii ticks in Saudi Arabia. MATERIALS AND METHODS: We collected 151 H. dromedarii ticks from 33 camels from 13 locations in Saudi Arabia. The second internal transcribed spacer (ITS2), cytochrome c oxidase subunit-1(COI), and 16S rRNA genes were used for single- and multi-locus sequence typing and phylogenetic analyses. H. dromedarii-borne Francisella was screened using the tul4 gene and 16S rRNA Francisella-specific primers followed by amplicon Sanger sequencing. RESULTS: Single-locus typing of ticks using ITS2, 16S rRNA, and COI genes yielded 1, 10, and 31 sequence types (ST), respectively, with pairwise sequence similarity of 100% for ITS2, 99.18-99.86% for COI, and 99.50-99.75% for 16S rRNA. COI sequence analysis indicated a lack of strict geographical structuration, as ST15 was found in both Saudi Arabia and Kenya. In contrast, multilocus sequence typing resolved 148 H. dromedarii ticks into 39 genotypes of ticks and three genotypes of FLEs. The ST2-FLE genotype was carried by the tick genotype ST35, while the ST1-FLE genotype and 41.89% of the ST3-FLE genotype were carried by the tick genotype ST32. Accordingly, there appeared to be no specialization of certain tick genotypes to harbor-specific FLE genotypes. CONCLUSION: For the 1st time, we have provided an overview of the population structure of H. dromedarii ticks and FLE strains. We found a low level of genetic diversity among FLEs and non-specialized circulation of FLEs among H. dromedarii ticks.

A glimpse of the bacteriome of Hyalomma dromedarii ticks infesting camels reveals human Helicobacter pylori pathogen.

INTRODUCTION: The tick Hyalomma dromedarii is predominant in camels of Saudi Arabia and harbor multiple pathogens causing disease in humans and animals. Knowing the bacterial community of ticks is crucial for surveillance of known and newly emerging pathogens. Yet, the bacteriome of H. dromedarii remain unexplored to date. METHODOLOGY: In a cross-sectional survey, we used V3-V4 region of 16S rRNA to characterize the bacteriome of 62 whole H. dromedarii tick samples collected from camels found in Hofuf city in Saudi Arabia. RESULTS: Sequencing results yielded 217 species incorporated into 114 genera, which in turn belong to the dominant phylum Proteobacteria (98%) followed by Firmicutes (1.38%), Actinobacteria (0.36%), Bacteroidetes (0.17%), meanwhile the phyla Cyanobacteria, Verrucomicrobia and unclassified bacteria were rarely detected. Francisella endosymbiont dominated the bacteriome of H. dromedarii ticks with average abundance of 94.37% and together with Salincoccus sp. accounted for 94.51% of the average sequences. The remaining bacteriome consisted of low abundance of potential pathogens and environmental bacteria. Of these pathogens, we found Helicobacter pylori in the tick H. dromedarii for the first time. Notably, Anaplasma, Ehrlichia and Rickettsia pathogens known to be found in H. dromedarii ticks were not detected. CONCLUSION: This first preliminary study advances our knowledge about the bacterial community of H. dromedarii ticks and provides a basis for pathogen surveillance and studying the influences of symbionts on vector competence. Presence of pathogens in ticks, raise concerns about potential transmission of these agents to humans or animals.

A one-step DNA PCR-based method for the detection of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media.

A simple, rapid, and sensitive direct colony polymerase chain reaction (PCR) method to detect Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media is described. M. tuberculosis is killed by treating it for 2 hours with 70% ethanol. Whole Mycobacterium cells inactivated by ethanol are added to a PCR mix that is designed to amplify the IS6110 insertion sequence. All 44 isolates tested were positive by this method. Our results show that PCR can be performed directly on bacterial colonies without the need for DNA extraction before PCR. Moreover, inactivation of M. tuberculosis before DNA amplification reduces the potential exposure of workers to viable M. tuberculosis. The exclusion of DNA extraction and inactivation of colonies before PCR provide a safe and low-cost preparatory technique for PCR reaction compared with expensive conventional extraction protocols that are based on chemical and enzymatic lysis, especially for countries with limited resources.

Chromosome and Megaplasmid Sequences of Borrelia anserina (Sakharoff 1891), the Agent of Avian Spirochetosis and Type Species of the Genus.

Sequences of the linear chromosome and plasmids of Borrelia anserina, the cause of avian spirochetosis of poultry, revealed a smaller genome than those of other Borrelia spp. transmitted by argasid ticks. Missing or disrupted genes included a dam methylase and those in the pathway for synthesis of phospholipids from glycerol.

Results of application of the ISPD guidelines to the management of peritoneal dialysis in a single center in Sudan.

The culture negative peritonitis in Sudan 2010 was 46% exceeding 20% of the recommended ISPD (International Society for Peritoneal Dialysis) guidelines. This study reports an update after applying the standard ISPD protocol. The routine method was replaced by ISPD protocol. The culture negative rate using the ISPD guidelines dropped from 46% in the year 2010, to 39% in the year 2011, to 5% in the 2012 and to zero percent in the year 2013. Bacterial and fungal species represent (86.76%) and (13.23%) of infection and most isolates showed low resistance rate to antibiotics. Touch contamination added significantly (p=0.0006) to the risk of contracting Peritonitis. The risk of contracting Peritonitis was 1.53 times higher in the group exposed by touch contamination. None of the other risk factors contributed significantly to Peritonitis. The study highlights the importance of implementing high hygiene practice.

Ubiquitous bacteria Borrelia crocidurae in Western African ticks Ornithodoros sonrai.

BACKGROUND: In West Africa, tick-borne relapsing fever is a neglected arthropod-borne infection caused by Borrelia crocidurae transmitted by the argasid tick Ornithodoros sonrai. From an epidemiological point of view, it is of interest to know whether some genotypes of the vector are specialized in carrying certain genotypes of the pathogen. FINDINGS: Thirty-five O. sonrai ticks collected in Mali, Senegal, Mauritania and Morocco confirmed to be B. crocidurae-infected, were genotyped by 16S rRNA gene sequencing. B. crocidurae was genotyped by Multispacer Sequence Typing. The 35 O. sonrai ticks grouped into 12 genotypes with strong geographical structuration. MST resolved the 35 B. crocidurae isolates into 29 genotypes with pairwise divergence of 0.09 - 1.56 % without strict geographical structuration as genotype ST22 was found in Mali, Senegal and Mauritania. There was no evidence of tick-borrelia specialization as one O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes. CONCLUSIONS: This report illustrates a non-specialized circulation of B. crocidurae borreliae within O. sonrai ticks in West Africa.

Genome Sequence of the Relapsing Fever Borreliosis Species Borrelia hispanica.

Borrelia hispanica is the etiological pathogen of tick-borne relapsing fever, transmitted to humans by infected Ornithodoros erraticus ticks. Here we present the 1,783,846-bp draft genome sequence, with an average G+C content of 28%. It has 2,140 open reading frames, 3 ribosomal RNAs, and 32 transfer RNAs.

African relapsing Fever borreliae genomospecies revealed by comparative genomics.

BACKGROUND: Relapsing fever borreliae are vector-borne bacteria responsible for febrile infection in humans in North America, Africa, Asia, and in the Iberian Peninsula in Europe. Relapsing fever borreliae are phylogenetically closely related, yet they differ in pathogenicity and vectors. Their long-term taxonomy, based on geography and vector grouping, needs to be re-apprised in a genomic context. We therefore embarked into genomic analyses of relapsing fever borreliae, focusing on species found in Africa. RESULTS: Genome-wide phylogenetic analyses group Old World Borrelia crocidurae, Borrelia hispanica, B. duttonii, and B. recurrentis in one clade, and New World Borrelia turicatae and Borrelia hermsii in a second clade. Accordingly, average nucleotide identity is 99% among B. duttonii, B. recurrentis, and B. crocidurae and 96% between latter borreliae and B. hispanica while the similarity is 86% between Old World and New World borreliae. Comparative genomics indicates that the Old World relapsing fever B. duttonii, B. recurrentis, B. crocidurae, and B. hispanica have a 2,514-gene pan genome and a 933-gene core genome that includes 788 chromosomal and 145 plasmidic genes. Analyzing the role that natural selection has played in the evolution of Old World borreliae species revealed that 55 loci were under positive diversifying selection, including loci coding for membrane, flagellar, and chemotaxis proteins, three categories associated with adaption to specific niches. CONCLUSION: Genomic analyses led to a reappraisal of the taxonomy of relapsing fever borreliae in Africa. These analyses suggest that B. crocidurae, B. duttonii, and B. recurrentis are ecotypes of a unique genomospecies, while B. hispanica is a distinct species.

Genome Sequence of the Asiatic Species Borrelia persica.

We report the complete genome sequence of Borrelia persica, the causative agent of tick-borne relapsing fever borreliosis on the Asian continent. Its genome of 1,784,979 bp contains 1,850 open reading frames, three ribosomal RNAs, and 32 tRNAs. One clustered regularly interspaced short palindromic repeat (CRISPR) was detected.

Relapsing fever borreliae in Africa.

The study of relapsing fever borreliae in Africa has long suffered from the use of non-specific laboratory tools for the direct detection of these spirochetes in clinical and vector specimens. Accordingly, Borrelia hispanica, Borrelia crocidurae, Borrelia duttonii, and Borrelia recurrentis have traditionally been distinguished on the basis of geography and vector and the unproven hypothesis that each species was exclusive to one vector. The recent sequencing of three relapsing fever Borrelia genomes in our laboratory prompted the development of more specific tools and a reappraisal of the epidemiology in Africa. Five additional potential species still need to be cultured from clinical and vector sources in East Africa to further assess their uniqueness. Here, we review the molecular evidence of relapsing fever borreliae in hosts and ectoparasites in Africa and explore the diversity, geographical distribution, and vector association of these pathogens for Africans and travelers to Africa.

Staphylococcus aureus subsp. anaerobius strain ST1464 genome sequence.

Staphylococcus aureus subsp. anaerobius is responsible for Morel's disease in animals and a cause of abscess in humans. It is characterized by a microaerophilic growth, contrary to the other strains of S. aureus. The 2,604,446-bp genome (32.7% GC content) of S. anaerobius ST1464 comprises one chromosome and no plasmids. The chromosome contains 2,660 open reading frames (ORFs), 49 tRNAs and three complete rRNAs, forming one complete operon. The size of ORFs ranges between 100 to 4,600 bp except for two ORFs of 6,417 and 7,173 bp encoding segregation ATPase and non-ribosomal peptide synthase, respectively. The chromosome harbors Staphylococcus phage 2638A genome and incomplete Staphylococcus phage genome PT1028, but no detectable CRISPRS. The antibiotic resistance gene for tetracycline was found although Staphylococcus aureus subsp. anaerobius is susceptible to tetracycline in-vitro. Intact oxygen detoxification genes encode superoxide dismutase and cytochrome quinol oxidase whereas the catalase gene is impaired by a stop codon. Based on the genome, in-silico multilocus sequence typing indicates that S. aureus subsp. anaerobius emerged as a clone separated from all other S. aureus strains, illustrating host-adaptation linked to missing functions. Availability of S. aureus subsp. anaerobius genome could prompt the development of post-genomic tools for its rapid discrimination from S. aureus.

Multiplex real-time PCR diagnostic of relapsing fevers in Africa.

BACKGROUND: In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples. METHODOLOGY/PRINCIPAL FINDINGS: We designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae. CONCLUSIONS/SIGNIFICANCE: The multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae.

Multispacer sequence typing relapsing fever Borreliae in Africa.

BACKGROUND: In Africa, relapsing fevers are neglected arthropod-borne infections caused by closely related Borrelia species. They cause mild to deadly undifferentiated fever particularly severe in pregnant women. Lack of a tool to genotype these Borrelia organisms limits knowledge regarding their reservoirs and their epidemiology. METHODOLOGY/PRINCIPAL FINDINGS: Genome sequence analysis of Borrelia crocidurae, Borrelia duttonii and Borrelia recurrentis yielded 5 intergenic spacers scattered between 10 chromosomal genes that were incorporated into a multispacer sequence typing (MST) approach. Sequencing these spacers directly from human blood specimens previously found to be infected by B. recurrentis (30 specimens), B. duttonii (17 specimens) and B. crocidurae (13 specimens) resolved these 60 strains and the 3 type strains into 13 species-specific spacer types in the presence of negative controls. B. crocidurae comprised of 8 spacer types, B. duttonii of 3 spacer types and B. recurrentis of 2 spacer types. CONCLUSIONS/SIGNIFICANCE: Phylogenetic analyses of MST data suggested that B. duttonii, B. crocidurae and B. recurrentis are variants of a unique ancestral Borrelia species. MST proved to be a suitable approach for identifying and genotyping relapsing fever borreliae in Africa. It could be applied to both vectors and clinical specimens.

Ovine clone ST1464: a predominant genotype of Staphylococcus aureus subsp. anaerobius isolated from sheep in Sudan.

BACKGROUND: The aim of the present study was to examine the phenotypic and genotypic relatedness of 17 Staphylococcus aureus subsp. anaerobius isolates recovered from sheep abscesses in Khartoum state, Sudan, during the period 2007-2008. METHODOLOGY: This sample was characterised using antibiogram typing, biochemical typing with the commercial PhenePlate system (PhP-CS) and multilocus sequence typing (MLST). RESULTS: Low levels of resistance were noted to the 11 antimicrobial agents tested. All the isolates corresponded to a single PhP type, and to a single, novel, multilocus sequence type, designated ST1464. CONCLUSION: These results demonstrate that the vast majority of cases of sheep abscess disease in Khartoum state are caused by a single novel clone of S. aureus subsp. anaerobius.