Biosensor for screening bacterial mercury methylation: example within the Desulfobulbaceae

Mercury methylation and demethylation processes govern the fate of methylmercury in aquatic ecosystems. Under anoxic conditions, methylation activity is mainly of biological origin and is often the result of sulfate-reducing bacteria. In this study, the use of a luminescent biosensor for screening methylmercury production was validated by exposing the reporter strain to methylating or non-methylating Desulfovibrio strains. The sensitivity of the biosensor to methylmercury was shown to depend on sulfate-reducing bacterial growth conditions. Bioluminescence was measured using 1–10 mM of sulfides. As the sulfide level increased, luminescence decreased by 40–70%, respectively. Nevertheless, assuming an average of 5 mM of sulfide produced during sulfate-reducing growth, a mercury methylation potential of over 4% was detected when using 185 nM of inorganic mercury. Due to technical limitations, mercury speciation has, to date, only been investigated in a small number of bacterial strains, and no consistent phylogenetic distribution has been identified. Here, the biosensor was further used to assess the Hg methylation capacities of an additional 21 strains related to the Desulfobulbaceae. Seven of them were identified as methylmercury producers. Cultivation procedures combined with bacterial biosensors could provide innovative tools to identify new methylator clades amongst the prokaryotes.     

A new genome of Acidithiobacillus thiooxidans provides insights into adaptation to a bioleaching environment

Acidithiobacillus thiooxidans is a sulfur oxidizing acidophilic bacterium found in many sulfur-rich environments. It is particularly interesting due to its role in bioleaching of sulphide minerals. In this work, we report the genome sequence of At. thiooxidans Licanantay, the first strain from a copper mine to be sequenced and currently used in bioleaching industrial processes. Through comparative genomic analysis with two other At. thiooxidans non-metal mining strains (ATCC 19377 and A01) we determined that these strains share a large core genome of 2109 coding sequences and a high average nucleotide identity over 98%. Nevertheless, the presence of 841 strain-specific genes (absent in other At. thiooxidans strains) suggests a particular adaptation of Licanantay to its specific biomining environment. Among this group, we highlight genes encoding for proteins involved in heavy metal tolerance, mineral cell attachment and cysteine biosynthesis. Several of these genes were located near genetic motility genes (e.g. transposases and integrases) in genomic regions of over 10 kbp absent in the other strains, suggesting the presence of genomic islands in the Licanantay genome probably produced by horizontal gene transfer in mining environments.     

Reclassification of the Caliciviridae into distinct genera and exclusion of hepatitis E virus from the family on the basis of comparative phylogenetic analysis

Summary.  Caliciviridae and Picornaviridae belong to the same subphylum and genera within Picornaviridae are well characterized. Until 1998, Caliciviridae included one genus Calicivirus, containing strains with distinct structural and genomic features. Phylogenetic analyses of capsid genes revealed five clusters within Caliciviridae corresponding to differences in genome organization. In order to determine to what taxonomic level these clusters correspond, genomic sequences of caliciviruses, picornavirus prototypes, and two togavirus strains were analyzed. Distance and maximum likelihood methods were used to estimate the phylogenetic relationships among strains. Analysis of the capsid gene revealed separation of five main clusters (Norwalk-like, and Sapporo-like human caliciviruses, hepatitis E virus, vesicular exanthem of swine-like, and lapine caliciviruses) and distances corresponding to those observed among picornavirus genera. Utilizing more conserved (presumed helicase and polymerase) regions for the analyses, only major groups of caliciviruses were separated with confidence, with distances also comparable to those separating picornavirus genera. Anal- ysis in these regions that included togavirus sequences moved HEV strains out of the calicivirus cluster. Our findings support the reclassification of caliciviruses into four genera. The phylogenetic position of hepatitis E virus, by analysis of non-structural genes, is outside of the caliciviruses, in an uncertain taxonomic position. Received October 22, 1999 Accepted January 13, 2000     

Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.     

Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.     

Genotyping of multi drug resistant Bacteroides fragilis group of clinical isolates from mangalore,south India

IntroductionBacteroides fragilis group, the most encountered anaerobic bacterium is emerging with resistance to antibiotics. This study explores the antibiogram and occurrence of resistance genes in isolates of B fragilis group from clinical samples.MethodIn this study the antimicrobial susceptibility test was done using commercially available E strip test and the results were recorded according to CLSI guidelines. Genotypic investigations were performed by conventional PCR to detect the target resistant genes.ResultsCeftriaxone, cefoxitin, clindamycin and imipenem were found to be the most resistant antimicrobials in E test method. Metronidazole has shown resistance in 7 strains in vitro while resistance nim genes were detected in 12 strains from 62 randomly selected isolates. Other resistance genes (cfiA, ermF and cepA) were expressed at 58%, 62.9% and 48.3% respectively, among these strains.ConclusionB fragilis group harbouring the resistant genes may not be fully expressed phenotypically. Hence, detection of these genes by PCR might be necessary for a pertinent conclusion.     

Bisulfite genomic sequencing to uncover variability in DNA methylation: Optimized protocol applied to human T cell differentiation genes

DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, is one of several epigenetic mechanisms that cells use to control gene expression. The importance of DNA methylation in a variety of biological processes (i.e., embryonic development, cellular proliferation and differentiation, chromosome stability) has led to a demand for a precise and efficient method to determine the exact DNA methylation status. Bisulfite genomic sequencing is regarded as a gold-standard technology for detection of DNA methylation as it provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine at single base-pair resolution. To optimize the final results of the bisulfite genomic sequencing protocol, numerous modifications have been explored and have significantly improved the sensitivity and accuracy of this procedure. The aim of this methodological report is to give an overview of the bisulfite genomic sequencing protocol, discussing the critical methodological aspects. Since we are interested in studying the methylation status of specific genes involved in T cell development, we applied the bisulfite genomic sequencing to the study of the CD8A T cell co-receptor gene to determine whether the CGIs of this gene were subjected to methylation in different types of tissues. The results show that CD8A gene is differentially methylated depending on the tissue. In conclusion, we described a bisulfite genomic sequencing protocol that can be successfully used for the quantitative analysis of CpG island methylation of specific genes.     

Helicobacter pylori Infection Introduces DNA Double-Strand Breaks in Host Cells

Gastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogen Helicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data that H. pylori itself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms of H. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening of H. pylori-infected human gastric biopsy specimens to identify candidate genes involved in H. pylori-induced host genomic instabilities. We found upregulation of ATM expression in vivo in gastric mucosal cells infected with H. pylori. Using gastric cancer cell lines, we confirmed that the H. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with both cag pathogenicity island (PAI)-positive and -negative strains, but the effect was more robust with cag PAI-positive strains. These results are consistent with the fact that infections with both cag PAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected with cag PAI-positive strains.     

Genomic diversity of two lineages of exfoliative toxin A-converting phages predominating in Staphylococcus aureus strains in the Czech Republic

We have isolated and characterized two distinct types of exfoliative toxin A (ETA)-converting bacteriophages originating from Staphylococcus aureus strains responsible for massive outbreaks of pemphigus neonatorum in the Czech Republic. Three induced phages designated as φB531, φB557 and φB122 were found to be capable of transferring the eta gene into the prophageless non-toxigenic S. aureus strain and converting it into an ETA producer. Comparisons of the phage sequences derived from 12 selected genes and 2 genomic segments (polymorphic P2 and conserved C4) revealed that φB531 and φB557 were identical each other, but φB122 differed from them in 5 gene sequences, the xis gene content and the virion protein profile. Thus, φB122 represents a new type of still undescribed ETA-converting phage. This study highlights not only the conclusive genomic diversity of eta gene-positive phages, but also their virulence implications in impetigo S. aureus strains.     

Comparative genomic analysis of the Dietzia genus: an insight into genomic diversity,and adaptation

Dietzia strains are widely distributed in the environment, presenting an opportunistic role, and some species have undetermined taxonomic characteristics. Here, we propose the existence of errors in the classification of species in this genus using comparative genomics. We performed ANI, dDDH, pangenome and genomic plasticity analyses better to elucidate the phylogenomic relationships between Dietzia strains. For this, we used 55 genomes of Dietzia downloaded from public databases that were combined with a newly sequenced. Sequence analysis of a phylogenetic tree based on genome similarity comparisons and dDDH, ANI analyses supported grouping different Dietzia species into four distinct groups. The pangenome analysis corroborated the classification of these groups, supporting the idea that some species of Dietzia could be reassigned in a possible classification into three distinct species, each containing less variability than that found within the global pangenome of all strains. Additionally, analysis of genomic plasticity based on groups containing Dietzia strains found differences in the presence and absence of symbiotic Islands and pathogenic islands related to their isolation site. We propose that the comparison of pangenome subsets together with phylogenomic approaches can be used as an alternative for the classification and differentiation of new species of the genus Dietzia.     

Towards a Molecular Definition of Enterohemorrhagic Escherichia coli (EHEC): Detection of Genes Located on O Island 57 as Markers To Distinguish EHEC from Closely Related Enteropathogenic E. coli Strains

Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx₁, stx₂, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains.     

Molecular characterization of the genome of duck enteritis virus

The genomic sequence of a strain of duck enteritis virus (DEV) was determined and analyzed in this study. The size of its genome is 158,091 bp in length and the genome is predicted to encode 78 putative proteins and resembles the members of the Alphaherpesvirinae in genomic organization and gene composition. The genome of the virus is composed of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region. Its genomic arrangement pattern (UL-IRS-US-TRS) corresponds to D-type herpesvirus and is consistent with the members of Varicellovirus and Iltovirus genera. Sequence analysis reveals that the genome of the virus contains 67 genes having homologs in most members of the Alphaherpesvirinae. Out of these genes, one gene has a homolog in cercopithecine herpesvirus 8 which is a virus of Betaherpesvirinae, and 5 genes have homologs in avian herpesviruses. Furthermore, the genome possesses three unique genes without homologs in any other herpesviruses. Like most members of the Alphaherpesvirinae, the genes in the UL region of its genome are well conserved, whereas the gene arrangement of IRS-US is similar to that of Marek's disease virus and equine herpesviruses 1. Therefore, our data based on the genomic analysis suggest that DEV represents an osculant taxonomic entity within the Alphaherpesvirinae.     

Isolation,characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria (Pseudovibrio sp.) and four with γ-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.     

Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ −10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.     

Desulfovibrio legallii Prosthetic Shoulder Joint Infection and Review of Antimicrobial Susceptibility and Clinical Characteristics of Desulfovibrio Infections

We describe a case of shoulder hemiarthroplasty infection with Desulfovibrio legallii. Antimicrobial susceptibilities of 36 Desulfovibrio isolates are presented. Metronidazole and carbapenems exhibited reliable activity, although piperacillin-tazobactam did not. Eleven previous cases of Desulfovibrio infection are reviewed; most arose from a gastrointestinal tract-related source.     

Aberrant DNA methylation as a diagnostic biomarker of diabetic embryopathy

PurposeMaternal diabetes is a known teratogen that can cause a wide spectrum of birth defects, collectively referred to as diabetic embryopathy (DE). However, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests to establish the diagnosis. Here, we explore the potential of DNA methylation as a diagnostic biomarker for DE and to inform disease pathogenesis.MethodsBisulfite sequencing was used to identify gene regions with differential methylation between DE neonates and healthy infants born with or without prenatal exposure to maternal diabetes, and to investigate the role of allele-specific methylation at implicated sites.ResultsWe identified a methylation signature consisting of 237 differentially methylated loci that distinguished infants with DE from control infants. These loci were found proximal to genes associated with Mendelian syndromes that overlap the DE phenotype (e.g., CACNA1C, TRIO, ANKRD11) or genes known to influence embryonic development (e.g., BRAX1, RASA3). Further, we identified allele-specific methylation (ASM) at 11 of these loci, within which 61.5% of ASM single-nucleotide variants are known expression quantitative trait loci (eQTLs).ConclusionsOur study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.     

Genome-wide analysis shows association of epigenetic changes in regulators of Rab and Rho GTPases with spinal muscular atrophy severity

Spinal muscular atrophy (SMA) is a monogenic disorder that is subdivided into four different types and caused by survival motor neuron gene 1 (SMN1) deletion. Discordant cases of SMA suggest that there exist additional severity modifying factors, apart from the SMN2 gene copy number. Here we performed the first genome-wide methylation profiling of SMA patients and healthy individuals to study the association of DNA methylation status with the severity of the SMA phenotype. We identified strong significant differences in methylation level between SMA patients and healthy controls in CpG sites close to the genes CHML, ARHGAP22, CYTSB, CDK2AP1 and SLC23A2. Interestingly, the CHML and ARHGAP22 genes are associated with the activity of Rab and Rho GTPases, which are important regulators of vesicle formation, actin dynamics, axonogenesis, processes that could be critical for SMA development. We suggest that epigenetic modifications may influence the severity of SMA and that these novel genetic positions could prove to be valuable biomarkers for the understanding of SMA pathogenesis.     

Emergence of Staphylococcus aureus strains resistant to pristinamycin in Sfax (Tunisia)

AimWe report the emergence of Staphylococcus aureus resistant to pristinamycin in Tunisia, and the characterization of the mechanisms of resistance to macrolides and streptogramins.Methods and resultsFive strains of S. aureus resistant to pristinamycin were recovered from the department of dermatology in a Tunisian university hospital from skin samples after oral use of pristinamycin between 2004 and 2007. Susceptibility testing showed that all isolates were resistant to quinupristin–dalfopristin (MIC = 4–32 mg/L), lincomycin, gentamicin, kanamycin, tobramycin, tetracycline and rifampin. One isolate was susceptible to erythromycin. All five strains were closely related after analysis by pulsed-field gel electrophoresis. erm(C) was amplified from three strains and erm(A) from one strain. vga and vat genes were amplified from all strains. None of the isolates carried the vgb gene. The vga and vat genes were typed as vga(B) and vat(B) by restriction profiles analysis after electrophoresis.ConclusionThis is the first report of clonal emergence of S. aureus resistant to pristinamycin carrying vga and vat genes in Tunisia. The role of selective pressure of pristinamycin use is certainly the main explanation of this emergence. So we must reduce the utilisation of this antibiotic for the treatment of cutaneous and bone infectious disease caused by multidrug resistant bacteria.