Bacterial Chromosomal DNA Research Articles

Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Study and Utilization of the K2 Mycobacteriophage Findley as an Antibacterial Agent

This project focuses on bacteriophages, viruses that infect host bacterial cells. When infected, the host bacterial cell will either enter the lytic or lysogenic phase. In the lytic cycle, the host cell will replicate phage DNA and create new phages. The cell will lyse to release the newly created phages. During lysogeny, the phage genome is integrated into the bacterial cell genome. New phages are not produced, and the host cell does not lyse. Specifically, this project focuses on mycobacteriophages, phages which infect bacteria of the genus Mycobacterium. In particular, we are looking for phages that will infect the host bacterium Mycobacterium tuberculosis, so that they might serve as a clinical therapy to fight tuberculosis infection. To do so, our goal is to genetically modify K2 phages, which are known to infect M. tuberculosis. I have focused on the K2 phage Findley, isolated by the SEA PHAGES program at Nyack College in Nyack, New York. Initially, we purified a Findley lysogen, which is a bacterial host cell with the Findley phage DNA incorporated into the bacterial cell's chromosomal DNA. In order for a phage to enter the lysogenic cycle, and for its DNA to be incorporated into a host genome, the phage must contain an integrase gene. The integrase gene determines where the phage DNA will be incorporated into the host genome, through a method called site‐specific recombination. We confirmed the existence of a Findley lysogen through a method of Polymerase Chain Reaction (PCR) which exploits site‐specific recombination. I used a series of primers to amplify certain segments of DNA at the exact points of recombination in the lysogen genome. The presence of these certain amplicons confirmed that lysogeny did occur, and that the integrase gene is functional in the Findley phage.After confirming the functionality of an integrase gene, the next goal of our project was genetic modification of the Findley phage. The goal of genetic modification is to change the phage so that it more efficiently infects and lyses the host Mycobacterium cells. To do so, we aim to knock out one of the genes responsible for the lysogen phase, the repressor gene. The repressor gene dictates that the bacterial cell be immune to further infection from the same phage and the cell becomes a lysogen. During lysogeny, the cell is not lysed. However, if a phage lacks this gene, it will only enter the lytic phase, making it a “killing” phage only. To construct this mutant phage, we performed a method of allelic exchange to delete the gene, called the BRED (Bacteriophage Recombineering with Electroporated DNA) method. The allelic exchange results in a specific portion of the phage genome being deleted. We designed a substrate DNA that is identical to regions of the phage genome, but with the specified gene missing. We used electrocompetent Mycobacterium smegmatis cells to perform the transformation. During the transformation, wild‐type phage DNA, the substrate DNA and electrocompetent M. smegmatis cells were electroporated so that the host bacterial cell will take both types of the DNA and recombine it. The M. smegmatis cell then produced the mutant phage during a lytic cycle. Plating the electroporated cells with host cells results in plaques that can be screened for phage with the desired deletion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length

Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Here, we show the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B) selectively controls placental expression of corticotropin-releasing hormone (CRH) that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5′ insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor expressed in placenta. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth.

Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice

Transgenic (Tg) mice containing bacterial artificial chromosome (BAC) DNA are widely used for gene expression analysis and gene therapy models because BAC transgenes provide gene expression at physiological levels with the same developmental timing as endogenous genes. To ensure correct interpretation of transgene functions, investigation of the genomic organisation and integration of the BAC transgene is required. Here, we describe a reliable method based on droplet digital PCR (ddPCR) and inverse PCR to estimate copy number, genomic organisation and insertion sites of BAC transgenes in the mouse genome. We generated BAC Tg mice containing fragments of BAC clone RP23-59P20. ddPCR and iPCR analysis showed that the transgene consisted of five fragments of the BAC clone containing the Mkrn3 gene region, and that the transgene was inserted into Bckdhb, homozygous deletion of which causes the maple syrup urine disease phenotype. The ddPCR method described here should prove useful for analysis of genomic organisation and integration of BAC transgenes.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

AbstractRapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype‐based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single‐molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point‐of‐care AST and strengthen global antibiotic stewardship.

592. High Quality Grade Plasmid DNA for Indirect Clinical Applications

Amongst other applications, our plasmid DNA is used in the GMP-compliant production of recombinant viruses, antibodies and RNA, where these are the active pharmaceutical ingredients (API) used in clinical trials. Here, it is not always necessary to produce the plasmid DNA under GMP as well, in order to use it for such applications. An alternative is the so-called High Quality Grade plasmid DNA which is highly purified and well-characterised and, hence, meeting the requirements of most regulating agencies.High Quality Grade plasmid DNA is produced in our facility based on a research cell bank (RCB) and the very effective, patented ccc Grade DNA technology. A number of quality controls, both to the cell bank and to the plasmid DNA product, ensure that the final result is a product designed especially for the intended application and that complies with the appropriate regulatory standards.PlasmidFactory's new facility for the production of this high purity plasmid DNA is now operating successfully in new, modern labs where plasmid DNA of the highest quality is being produced in accordance with the EMEA guideline CHMP/BWP/2458/03. To ensure product safety, substances of animal origin are not used at any stage of the entire process, guaranteeing maximum possible product purity by reliable exclusion of contaminants such as bacterial chromosomal DNA or damaged plasmids. Only one plasmid is produced in each area - different plasmids are not produced in parallel in the same lab.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Modeling the Binding of H-NS to DNA

Bacterial chromosomal DNA is organized within a structure called the nucleoid, which is distinctly different from the rest of the cytoplasm. Bacteria have a number of nucleoid-associated proteins that influence the organization of the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS can bridge DNA by binding to two separate DNA duplexes, or shield the DNA by binding to distant sites on the same duplex, depending on external conditions. H-NS occurs in Gram-negative enterobacteria and silences genes involved in bacterial virulence and antibiotic resistance.

Pulsed-field gel electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides.

Pulsed-field gel electrophoresis (PFGE) is a technique using alternating electric fields to help the separation of high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and of applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragment. This method is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.

High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis?

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

Copy Number Abnormalities Of The Interferon Regulatory Factor-4 (IRF4) Gene Are Associated With IRF4/MUM1 Expression In Peripheral T-Cell Lymphomas

BackgroundPeripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with poor overall survival rates. The genetics of PTCLs are poorly understood. We have described t(6;14)(p25.3;q11.2) translocations in PTCLs involving the T-cell receptor-alpha (TRA@) and interferon regulatory factor-4 (IRF4) genes and overexpressing IRF4 protein (also called MUM1). IRF4/MUM1 is a lymphoid transcription factor involved in differentiation and growth, the expression of which is tightly regulated in normal T cells. However, nearly half of PTCLs constitutively express IRF4/MUM1 and this expression is associated with adverse overall survival in several PTCL subtypes. Because IRF4/TRA@ translocations are rare among PTCLs (<1%), we investigated the presence of other genetic abnormalities of IRF4 in PTCLs and examined their association with IRF4/MUM1 protein expression. MethodsStudies were performed on paraffin tissue sections of PTCL specimens diagnosed by standard WHO criteria. IRF4/MUM1 immunohistochemical staining (IHC) was performed using the MUM1p antibody and considered positive if nuclear staining was present in >30% of tumor cell nuclei. Fluorescence in situ hybridization (FISH) was performed using breakapart probes constructed from bacterial artificial chromosome DNA that hybridized to loci flanking the IRF4 locus on 6p25.3. Cases with 3 or more fusion signals were considered to have extra copies of the IRF4 gene. In a subset of cases, PCR and Sanger sequencing was performed on DNA extracted from frozen PTCL tumor tissue with primer sets that amplified exons 1 to 10 of the IRF4 gene. Data were analyzed using the chi-square test or t test. ResultsPTCLs from 277 patients (175 M:102 F; median age, 61 y) were examined by both IHC and FISH. The subtype distribution was: PTCL, not otherwise specified (NOS), 93; anaplastic large cell lymphoma (ALCL), 89; angioimmunoblastic T-cell lymphoma (AITL), 27; cutaneous T-cell lymphoma (CTCL), 43; and cytotoxic PTCL, 25. IRF4/MUM1 was positive in 116 cases (42%). Extra copies of IRF4 were identified in 18 PTCLs (7%), 83% of which were IRF4/MUM1 protein-positive. In contrast, 39% of cases without extra copies of IRF4 were IRF4/MUM1 protein-positive (p=0.0002). The mean percentage of tumor cells with IRF4/MUM1 staining was 69% in cases with extra copies of IRF4, compared to 30% in cases without extra copies (p=0.0001). Extra copies of IRF4 were seen in 13% of ALCLs, 4% each of PTCLs, NOS and cytotoxic PTCLs, 2% of CTCLs, and 0% of AITLs (p=0.02). Two IRF4/MUM1-positive cases had an IRF4/TRA@ translocation (1%). We selected 30 IRF4/MUM1-positive PTCLs for Sanger sequencing. In this subset, we identified a previously undescribed non-synonymous variant in exon 5 (S309R) in 1 case (3%) without an extra copy of IRF4. ConclusionsExtra copies of IRF4 are seen in ∼7% of PTCLs, are significantly associated with IRF4/MUM1 expression, and are preferentially observed in ALCLs. Both IRF4/TRA@ translocations and IRF4 mutations are rare in PTCLs. These data suggest that genetic abnormalities of the IRF4 gene – specifically extra copies of IRF4 – may contribute to IRF4/MUM1 expression in some PTCLs. Since IRF4/MUM1 expression is seen in ∼42% of PTCLs, these data also imply the existence of other regulatory mechanisms. Characterizing these mechanisms may be important clinically, since IRF4/MUM1 has been proposed as a therapeutic target in human cancers but drugs that directly inhibit its function are currently not available. Disclosures:No relevant conflicts of interest to declare.

Integration Host Factor of Mycobacterium tuberculosis, mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.

Standard Reference Material 2366 for Measurement of Human Cytomegalovirus DNA

Human cytomegalovirus (CMV), classified as human herpesvirus 5, is ubiquitous in human populations. Infection generally causes little illness in healthy individuals, but can cause life-threatening disease in those who are immunocompromised or in newborns through complications arising from congenital CMV infection. An important aspect in diagnosis and treatment is to track circulating viral load with molecular methods, particularly with quantitative PCR. Standardization is vital, because of interlaboratory variability (due in part to the variety of assays and calibrants). Toward that end, the U.S. National Institute of Standards and Technology produced a Standard Reference Material 2366 appropriate for establishing metrological traceability of assay calibrants. This standard is composed of CMV DNA (Towne(Δ147) bacterial artificial chromosome DNA). Regions of the CMV DNA that are commonly used as targets for PCR assays were sequenced. Digital PCR was used to quantify the DNA, with concentration expressed as copies per microliter. The materials were tested for homogeneity and stability. An interlaboratory study was conducted by Quality Control for Molecular Diagnostics (Glasgow, UK), in which one component of SRM 2366 was included for analysis by participants in a CMV external quality assessment and proficiency testing program.

Gene Delivery Using Helper Virus‐Free HSV‐1 Amplicon Vectors

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only a very small percentage of the 152-kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication-defective mutants of HSV-1 or by replication-competent, but packaging-defective, HSV-1 genomes. Sets of cosmids that overlap and represent the entire HSV-1 genome can form, via homologous recombination, circular replication-competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid or bacterial artificial chromosome (BAC) DNA into 2-2 cells by cationic liposome-mediated transfection and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid and BAC DNA and determining the titers of amplicon stocks.

Single Molecule Live Cell Millisecond Fluorescence Imaging of Bacterial Condensins

The MukBEF protein complex in Escherichia coli is a condensin-like system that binds to bacterial chromosomal DNA. Using bespoke slimfield millisecond fluorescence imaging we were able to visualize the three MukB, E and F protein components in functional complexes in single live cells, both separately as genomic YPet fusions and multiply as dual-label GFP/mCherry mutants and obtain in vivo estimates for the stoichiometry of these components using step-wise photobleaching of fluorescent proteins, precise down to the level of single molecules. This indicates a predominately tetrameric complex of B:E:F = 4:4:2 suggesting that the DNA-bound MukBEF in the live cell is in its ATP-bound state, and which is highly dynamic in the living cell with focal complexes undergoing molecular turnover over a time scale of tens of seconds. The dynamic localization observed is consistent with a spontaneous oscillation-type model which may be used in the essential positioning of replicated DNA prior to successful DNA segmentation into newly divided cells.

Simple, fast, tissue‐specific bacterial artificial chromosome transgenesis in Xenopus

We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.

Confirmation of HIV-like sequences in respiratory tract bacteria of Cambodian and Kenyan HIV-positive pediatric patients

SummaryBackgroundBacteria and yeasts isolated from respiratory tracts of 39 Cambodian and 28 Kenyan HIV-positive children were tested for the presence of HIV-1 sequences.Material/MethodsBacteria and yeasts from the respiratory tract (nose, pharyngeal swabs) were isolated from 39 Cambodian and 28 Kenyan HIV-positive children. Bacterial chromosomal DNA was prepared by standard protocol and by Qiagen kit. The PCR specific for HIV sequences was carried out using HIV-1-specific primers. The analysis was performed by colony and dot-blot hybridization using HIV-1-specific primers which represent gag, pol and env genes of the virus. The sequencing of some PCR products was performed on the ABI 373 DNA Sequencer.ResultsThe majority of microbes were characterized as Staphylococcus aureus, Klebsiella pneumoniae, and resp. Candida albicans. In some cases E. coli, Streptococcus pyogenes, Proteus mirabilis and Candida tropicalis were identified. Bacteria of 16 Cambodian (41%) and 8 Kenyan (31%) children were found to be positive in colony and dot-blot DNA hybridization. By the sequencing of PCR products synthesized on the template of patients’ bacterial DNA using primers 68;69 for env HIV-1 gene, homology of greater than 90% with HIV-1 isolate HXB2 (HIVHXB2CG) was revealed.ConclusionsBacteria and yeasts from the respiratory tract of 41% of Cambodian and 31% of Kenyan HIV-positive children bear HIV-like sequences. The role of bacteria in the HIV disease process is discussed.