Including Laboratory Strains Research Articles

A common anti-cytomegalovirus drug, ganciclovir, inhibits HIV-1 replication in human tissues ex vivo.

Cytomegalovirus (CMV) is a common HIV-1 copathogen. Since CMV infection is an important contributor to immune activation, the driving force of HIV disease, an anti-CMV strategy might be beneficial to HIV-infected patients. Shin et al. (J Acquir Immune Defic Syndr 2014; 65:251-258) reported that anti-CMV therapy with valganciclovir in coinfected individuals results in a decrease of HIV viral load that is not accompanied by a decrease of immune activation. This suggests an alternative mechanism for HIV inhibition other than suppression of CMV-mediated inflammation. We evaluated the anti-HIV activity of ganciclovir (GCV), the active form of valganciclovir, on HIV replication in human tissues ex vivo. We show that GCV has a direct suppressive activity on HIV replication in human tissues ex vivo, including laboratory strains, drug-resistant and primate HIV-1 isolates. We deciphered the mechanism of this inhibition and showed that GCV-TP is incorporated in the nascent DNA chain and acts as a delayed chain terminator. Our results suggest that anti-CMV strategy using valganciclovir in HIV-1-infected individuals may reduce HIV-1 viral load not only indirectly by decreasing CMV-mediated immune activation but also directly by inhibiting HIV-1 reverse transcriptase.

M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures

Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5- and X4–tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission.

Molecular characterization of antibiotic resistance in Pseudomonas and Aeromonas isolates from catfish of the Mekong Delta, Vietnam

A collection of 116 motile Pseudomonas spp. and 92 Aeromonas spp. isolated from 15 Vietnamese intensive catfish farms was analyzed to examine the molecular antibiotic resistance characteristics and the transferability of resistance markers within and between species. High levels of resistance to ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, chloramphenicol, and nitrofurantoin were observed. The percentage of multiple drug resistance of Pseudomonas spp. and Aeromonas spp. isolates was 96.6% and 61.9%, respectively. The multiple antibiotic resistance (MAR) index mean values of 0.457 and 0.293 of Pseudomonas and Aeromonas isolates, respectively, indicated that these isolates were exposed to high risk sources of contamination where antibiotics were commonly used. Approximately 33% of Pseudomonas spp. and 28% of Aeromonas spp. isolates from catfish contained class 1 integrons, but no class 2 integrons were detected. Several common resistance genes including aadA, dfrA and catB were harbored in class 1 integrons. Large plasmids (>55kb) were frequently detected in 50% and 71.4% of the plasmids extracted from Pseudomonas and Aeromonas isolates, respectively. Conjugation and transformation experiments demonstrated the successful transfer of all or part of the resistance phenotypes of catfish isolates to the recipient strains, including laboratory strains and strains isolated from this study. These results highlight the likely role of catfish bacteria as a reservoir of antibiotic resistant, Gram-negative bacteria harboring a pool of mobile genetic elements that can readily be transferred intra- and interspecies. To our knowledge, this is the first report on molecular characterization of antibiotic resistance of bacteria isolated from catfish in Vietnam.

Dcm‐mediated cytosine DNA methylation is conserved in Escherichia coli and influences the expression of ribosomal protein genes

In Escherichia coli, cytosine DNA methylation is catalyzed by the Dcm (DNA cytosine methylase) protein and occurs at the second cytosine in the sequence 5′CCWGG3′. Although the presence of cytosine DNA methylation was reported over 35 years ago, the biological role of 5‐methylcytosine in E. coli remains unclear. There is data indicating that Dcm can protect the genome against attack by a restriction enzyme that cleaves the same sequence, yet DNA methyltransferases in eukaryotes often influence gene expression. In order to gain insight into the potential roles of cytosine DNA methylation in E. coli, we: (a) screened 162 strains including laboratory strains, pathogens, and recently isolated environmental samples for the presence of the full‐length dcm gene using the polymerase chain reaction; (b) examined the same strains for the presence of 5‐methylcytosine at 5′CCWGG3′ sites using a restriction enzyme isoschizomer digestion assay; and (c) quantified the levels of 5‐methyl‐2′‐deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm‐mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5‐methylcytosine ranges from 0.8–1.2% of the cytosines. We also tested the hypothesis that Dcm influences gene expression. Gene expression in wild‐type and dcm knockout cells was compared via qPCR. We focused on ribosomal protein genes, as they have been previously demonstrated to contain numerous 5′CCWGG3′ sites. We demonstrate that Dcm represses expression of ribosomal protein genes during stationary phase, and this may explain the ubiquitous presence of this DNA modification pathway. Support for this work was provided by the Geneseo Foundation and NIH grant R15AI074035‐01.

PHYLOGENY OFSPIROGYRAANDSIROGONIUM(ZYGNEMATOPHYCEAE) BASED ONRBCL SEQUENCE DATA1

DNA sequence data were obtained for the gene encoding the large subunit of RUBISCO (rbcL) from 26 strains ofSpirogyraand seven ofSirogonium, using as outgroups 10 genera in the Zygnematales and Desmidiales (Closterium,Cosmarium,Cylindrocystis,Gonatozygon,Mesotaenium,Netrium,Penium,Zygnema,Zygnemopsis,Zygogonium). Sequence data were analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI), with bootstrap replication (MP, ML) and posterior probabilities (BI) as measures of support. MP, ML, and BI analyses of therbcL data strongly support a single clade containingSpirogyraandSirogonium. TheSpirogyrataxa are monophyletic, with the exception ofSpirogyra maxima(Hassall) Wittrock, which is nested within a clade withSirogoniumand shares with them the characters of loosely spiraled chloroplasts (<1 complete turn per cell) and anisogamy of gametangial cells;S. maximadiffers fromSirogoniumin displaying well‐defined conjugation tubes rather than a tubeless connection involving bending (genuflection) of filaments. The ML and BI analyses place thisSirogonium/Spirogyra maximaclade sister to the remainingSpirogyra. Morphological differences among strains ofSpirogyragrouped together on the basis ofrbcL data, including laboratory strains derived from clonal cultures (Spirogyra communis,S. pratensis), indicate that some characters (filament width, chloroplast number) used in the traditional taxonomy of this group are poor measures of species identity. However, some characters such as replicate end walls and loose spiraling of chloroplasts may be synapomorphies forSpirogyraclades.

Mycobacterial growth and sensitivity to H2O2 killing in human monocytes in vitro.

The intracellular growth and susceptibilities to killing by H2O2 in cultured human monocytes of a number of mycobacterial species including laboratory strains and clinical isolates of Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin (BCG) and a clinical isolate of Mycobacterium avium-M. intracellulare were examined. The clinical isolate of M. avium-M. intracellulare did not replicate in freshly explanted monocytes (generation time of >400 h); BCG replicated with a generation time of 95 h, and M. tuberculosis strains CDC551, H37Rv, and H37Ra replicated with generation times of 24, 35, and 37 h, respectively, during the 4-day growth assay. When cultured in monocytes for 4 days, the mycobacteria were variably sensitive to H2O2-induced killing. A positive correlation between the generation time and percent killing of intracellular bacilli was observed. By comparison, mycobacterial strains were similarly sensitive to H2O2 treatment in cell-free culture media and in sonicated cell suspensions. Using a number of inhibitors of reactive oxygen intermediates we determined that other than catalase the inhibitors tested did not affect H2O2-induced killing of intracellular mycobacteria. Our studies suggest that the killing of mycobacteria growing in human monocytes in vitro by the addition of exogenous H2O2 is dependent on the susceptibility to a peroxide-induced killing pathway as well as on the intracellular growth rate of the mycobacteria.

Anti-Human Immunodeficiency Virus Type 1 Activity of R-95288, a Phosphodiester Hexadeoxyribonucleotide Modified by Dibenzyloxybenzyl and Hydroxyethyl Residues at the 5′- and 3′-Ends

The phosphodiester hexadeoxyribonucleotide R-95288 is a potent anti-human immunodeficiency virus type 1 (HIV-1) agent in vitro which consists or a TGGGAG nucleoside sequence with dibenzyloxybenzyl and hydroxyethyl substituents at the 5′- and 3′-ends, respectively. In this study, the antiviral activity of R-95288 against various strains of HIV-1 in vitro was assessed and its mechanism of action was analysed. R-95288 inhibited replication of all strains of HIV-1 used including laboratory strains with the syncytium-inducing (SI) phenotype and clinical isolates with both SI and non-SI (NSI) phenotypes. The 50% inhibitory concentrations (IC50s) were 0.62–18 μg mL−1(0.21–6.2 μM). R-95288 inhibited the binding and fusion of HIV-1-infected T cells with CD4+cells. In addition, R-95288 specifically blocked the binding of monoclonal antibodies, recognizing the anti-V3 loop or the CD4-binding site of the virus envelope glycoprotein gp120. Furthermore, the target site of R-95288 within the V3 loop was found in the putative heparin-binding region by binding inhibition assays using various anti-V3 loop antibodies. These results suggest that R-95288 can inhibit various strains of HIV-1, possibly by specific interaction with gp120.

Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates

Murine bone marrow-derived macrophages were infected with a panel of virulent isolates of Mycobacterium tuberculosis including laboratory strains Erdman and H37Rv and various clinical isolates in order to determine the sensitivity of each of these strains to the antimycobacterial activities of macrophage-generated reactive nitrogen intermediates (RNI). All of the M. tuberculosis strains grew in murine bone marrow-derived macrophages; however, gamma interferon-primed macrophages limited the initial growth of intracellular bacilli. Some of the mycobacterial strains, including Erdman, were killed over the first 4 days of infection, as evidenced by significant decreases in the number of viable intracellular bacilli determined by a CFU assay. Other mycobacterial strains were not killed during this same period, and some isolates, including CSU 24 and CSU 31, grew steadily in activated macrophages. The accumulation of nitrite on infected monolayers was measured, and it was found that inhibitory levels of RNI did not vary among infections with the different strains. Nitrite tolerance was determined in a cell-free system for each of the strains in order to compare susceptibilities of the strains to RNI. All of the strains tested were killed by levels of RNI generated by the acidification of 10 mM NaNO2 to pH 6.5 or 5.5, and the strains exhibited a range of tolerance to lower concentrations of RNI. No correlations were observed between such cell-free RNI tolerances and the capacity of bacilli to resist macrophage RNI-mediated killing. These results indicate that under stringent conditions, RNI can kill M. tuberculosis, but that under less harsh, more physiological conditions, the effects of RNI range from partial to negligible inhibition.

Detection ofActinomycesSpecies Using Nonradioactive Riboprobes Coupled with Polymerase Chain Reaction

We have been focusing our attention on the detection and identification of oral bacteria which are frequently associated with periodontal disease. In previous studies,Actinomycesspecies-specific riboprobes were generated and used to identify this microorganism. However, problems lie in the low sensitivity of this method. We have developed a novel system for the detection ofActinomycesspecies using nonradioactive riboprobes coupled with polymerase chain reaction (PCR) in this study. This system employs two procedures; initially, DNA fragments specific for the target microorganism are amplified by PCR, and these specific fragments are further hybridized with nonradioactive riboprobes. PCR analysis using chromosomal DNA isolated fromActinomycesspecies including laboratory strains, clinical isolates, andActinomyces naeslundii(ATCC 12104) indicated the presence of the predicted common 756-bp fragment, a portion of the sialidase gene. These amplified DNA fragments were effectively visualized by hybridization with the digoxigenin-labeled riboprobes corresponding to the internal region of the amplified sialidase gene. With this system, approximately three orders of magnitude less chromosomal DNA was sufficient for the detection of specific microorganisms compared to the conventional riboprobe systems.

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.

Serotype protein agglutinogens of Neisseria gonorrhoeae.

The distribution among Neisseria gonorrhoeae strains of the serotype protein agglutinogens (SPA) carried by the laboratory strains VII, V, and 8551 was studied. The bacterial outer membrane was isolated, treated with periodate, and used for immunization of rabbits. Each antiserum was cross-absorbed with both of the heterologous strains and examined by a bacterial agglutination test against 42 other gonococcal isolates, including laboratory strains and recent clinical isolates. In all, 36 of the isolates could be serotyped, 23 of which carried SPA VII, six SPA V, three SPA 8551, and four both of the SPAs V and 8551. Antiserum was prepared in a similar way against each of the six strains that could not be serotyped. These antisera showed varying patterns of cross-reactivity, depending on the strains used for cross-absorption.