Pharmacodynamics of ClpP-Activating Antibiotic Combinations against Gram-Positive Pathogens.

It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96 h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52 h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.

Beggiatoa leptomitoformis sp. nov., the first freshwater member of the genus capable of chemolithoautotrophic growth.

A strain of filamentous sulfur bacteria was isolated from freshwater spring contaminated with residential and agricultural wastewater in Moscow region, Russia. According to the results of phylogenetic analysis, strain D-402T belonged to the genus Beggiatoa within the family Beggiatoaceae of the class Gammaproteobacteria. Within the genus Beggiatoa, strain D-402T was most closely related to Beggiatoa alba strains. Strain D-402T had a DNA G+C content 42.1 mol%. The DNA-DNA hybridization value between strain D-402T and Beggiatoa alba strain B15LD was 33 %. Predominant fatty acids were C18 : 1 (46.1 and 53.3 %), C16 : 0 (15.5 and 16.2 %) and C16 : 1 (32.9 and 25.4 %) for strains D-402T and B15LD, respectively. In contrast to known representatives of Beggiatoa, strain D-402T was capable of chemolithoautotrophic growth with sulfide and thiosulfate as electron donors. Oxidation of sulfide and thiosulfate was accompanied by deposition of sulfur globules within the cells. Strain D-402T was capable of heterotrophic growth. The strain was capable of using different organic compounds, sulfur compounds and hydrogen as electron donors. Based on these observations, strain D-402T is considered as a representative of a species Beggiatoa leptomitoformis sp. nov. of the genus Beggiatoa. The type strain is D-402T (=DSM 14946T=UNIQEM U 779T).

Spirochaeta sinaica sp. nov., a halophilic spirochaete isolated from a cyanobacterial mat.

A strain of free-living obligately anaerobic, halophilic spirochaete, SLT, was isolated from a sample of a cyanobacterial mat of the hypersaline Solar Lake, Sinai shore. The strain had motile helical cells, 0.35-0.40 × 6-10 µm. Strain SLT exhibited high resistance to NaCl among known halophilic spirochaetes growing at NaCl concentrations from 2 to 12% (optimum growth at 7%). The strain grew at temperatures from 10 to 32 °C (optimum at 28 °C) and pH from 6 to 8.5 (optimum at pH 7.0-7.5). Carbohydrates, but not alcohols, organic acids or nitrogenous compounds (peptone, yeast extract and amino acids), were used as energy substrates for growth. Ethanol, acetate, lactate, H2 and CO2 were the products of glucose fermentation. Sulfide was produced in the presence of S0 or thiosulfate in the medium. The DNA G+C content was 44.7 mol%. Based on 16S rRNA gene sequence analysis, strain SLT clustered within the genus Spirochaeta, exhibiting 94.2 and 93.7% similarity with its closest relatives, Spirochaeta bajacaliforniensis DSM 160554T and Spirochaeta smaragdinae DSM 11293T, respectively; similarity with other species did not exceed 86%. The phenotypic and chemotaxonomic characteristics of the strain, as well as the results of phylogenetic analysis support the classification of strain SLT as representing a novel species of the genus Spirochaeta, for which the name Spirochaeta sinaica sp. nov. is proposed. The type strain is SLT ( = DSM 14994 = NIQEM U 783).

In vitro and in vivo activities of HPi1, a selective antimicrobial against Helicobacter pylori.

A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 µg/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.

Spirochaeta perfilievii sp. nov., an oxygen-tolerant, sulfide-oxidizing, sulfur- and thiosulfate-reducing spirochaete isolated from a saline spring.

A novel strain of fermenting, aerotolerant, chemo-organoheterotrophic spirochaete designated P(T) was isolated from a sulfur 'Thiodendron' mat in a saline spring at the Staraya Russa resort (Novgorod Region, Russia). Cells of strain P(T) exhibited a helical shape. The spirochaete required sulfide in the growth medium and was able to oxidize it non-enzymically to elemental sulfur via the interaction of H(2)O(2) with sulfide and deposit it in the periplasmic space. Growth occurred at 4-32 °C (optimum at 28-30 °C), pH 6.0-8.5 (optimum pH 7.0-7.5), and in 0.1-1 M NaCl (optimum 0.35 M). The isolate used several sugars and polysaccharides as carbon or energy sources but did not use peptides, amino acids, organic acids or alcohols. The products of glucose fermentation were formate, acetate, ethanol, pyruvate, CO(2) and H(2). The genomic DNA G+C content was 41.7 mol%. 16S rRNA gene sequence analysis showed that strain P(T) fell within a group of species in the genus Spirochaeta, including Spirochaeta litoralis, S. isovalerica and S. cellobiosiphila, with which it shared less then 89 % sequence similarity. On the basis of its morphology, physiology and other phenotypic properties, as well as its phylogenetic position, the new isolate is considered to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta perfilievii sp. nov. is proposed. The type strain is P(T) (=DSM 19205(T) =VKM B-2514(T)).

The Discovery and Development of Thienopyrimidines as Inhibitors of Helicobacter pylori That Act through Inhibition of the Respiratory Complex I.

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship (SAR) of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified H. pylori's respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.

A trap for in situ cultivation of filamentous actinobacteria.

The approach of growing microorganisms in situ, or in a simulated natural environment is appealing, and different versions of it have been described by several groups. The major difficulties with these approaches are that they are not selective for actinomycetes - a group of gram-positive bacteria well known as a rich source of antibiotics. In order to efficiently access actinomycetes, a trap for specifically capturing and cultivating these microorganisms in situ has been developed, based on the ability of these bacteria to form hyphae and penetrate solid environments. The trap is formed by two semi-permeable membranes (0.2-0.6 microm pore-size bottom membrane and 0.03 microm pore-size top membrane) glued to a plastic washer with sterile agar or gellan gum inside. The trap is placed on top of soil, and filamentous microorganisms selectively penetrate into the device and form colonies. Decreasing the size of the pores of the lower membrane to 0.2 microm restricted penetration of fungi. The trap produced more filamentous actinobacteria, and a higher variety of them, as compared to a conventional Petri dish cultivation from the same soil sample. Importantly, the trap cultivation resulted in the isolation of unusual and rare actinomycetes.

TM7 detection in human microbiome: Are PCR primers and FISH probes specific enough?

TM7 appears important and omnipresent because it is repeatedly detected by molecular techniques in diverse environments. Here we report that most of primers and FISH probes thought to be TM7-specific do hybridize with multiple species from oral and vaginal cavity. This calls for re-examination of TM7 distribution and abundance.

Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2.

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin.

Siderophores from neighboring organisms promote the growth of uncultured bacteria.

The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Proposal of Giesbergeria voronezhensis gen. nov., sp. nov. and G. kuznetsovii sp. nov. and reclassification of [Aquaspirillum] anulus, [A.] sinuosum and [A.] giesbergeri as Giesbergeria anulus comb. nov., G. sinuosa comb. nov. and G. giesbergeri comb. nov., and [Aquaspirillum] metamorphum and [A.] psychrophilum as Simplicispira metamorpha gen. nov., comb. nov. and S. psychrophila comb. nov.

Five Gram-negative, motile, spiral-shaped strains were isolated from a sulfide spring (D-412T), active sludge of wastewater (D-419T, D-420, D-424) and industrial wastewater (D-416). Comparative 16S rRNA gene sequence analysis showed that the isolates belong to the family Comamonadaceae, within the class Betaproteobacteria, but fall into a distinct cluster. On the basis of phenotypic, chemotaxonomic and phylogenetic data, a new genus, Giesbergeria gen. nov., is proposed, including five species. The type species of the genus is Giesbergeria voronezhensis sp. nov. (type strain D-419T = DSM 12825T = CIP 107340T = VKM B-2350T) and other novel members of the genus are Giesbergeria kuznetsovii sp. nov. (type strain D-412T = DSM 12827T = VKM B-2352T), Giesbergeria giesbergeri comb. nov. (basonym Aquaspirillum giesbergeri), Giesbergeria sinuosa comb. nov. (basonym Aquaspirillum sinuosum) and Giesbergeria anulus comb. nov. (basonym Aquaspirillum anulus). Using the same criteria, isolate D-416 (= DSM 12826) was identified as a strain of [Aquaspirillum] metamorphum. Strain D-416, the type strain of [A.] metamorphum and the type strain of [Aquaspirillum] psychrophilum form a distinct cluster within the family Comamonadaceae (97-97.2% 16S rRNA gene sequence similarity) and share phenotypic and chemotaxonomic properties. Therefore, it is proposed that these strains are reclassified as members of a new genus, Simplicispira gen. nov., as Simplicispira metamorpha comb. nov. (the type species) and Simplicispira psychrophila comb. nov., respectively.

Okibacterium fritillariae gen. nov., sp. nov., a novel genus of the family Microbacteriaceae.

Okibacterium fritillariae gen. nov., sp. nov. (type strain VKM Ac-2059T = IFO 16404T) is proposed for aerobic, oxidase- and catalase-positive, coryneform bacteria isolated from seeds of Fritillaria ruthenica Wikstr. and Clematis recta L. Strains of the new genus form a distinct branch within the phylogenetic cluster composed of members of the family Microbacteriaceae and are characterized by B-type peptidoglycan containing amino acids glycine, glutamate, homoserine, alanine and lysine, the glycolyl type of muramic acid, the major menaquinones MK-10 and MK-11, the principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, and a DNA G+C content of approximately 67 mol %.

A novel mannitol teichoic acid with side phosphate groups of Brevibacterium sp. VKM Ac-2118.

The cell wall of Brevibacterium sp. VKM Ac-2118 isolated from a frozen (mean annual temperature -12 degrees C) late Pliocene layer, 1.8-3 Myr, Kolyma lowland, Russia, contains mannitol teichoic acid with a previously unknown structure. This is 1,6-poly(mannitol phosphate) with the majority of the mannitol residues bearing side phosphate groups at O-4(3). The structure of the polymer was established by chemical methods, NMR spectroscopy, and MALDI-TOF mass spectrometry.