Modifying gut integrity and microbiome in children with severe acute malnutrition using legume-based feeds (MIMBLE): A pilot trial.

Case fatality among African children with severe acute malnutrition remains high. We report a 3-arm pilot trial in 58 Ugandan children, comparing feeds targeting disordered gastrointestinal function containing cowpea (CpF, n = 20) or inulin (InF, n = 20) with conventional feeds (ConF, n = 18). Baseline measurements of gut permeability (lactulose:mannitol ratio 1.19 ± SD 2.00), inflammation (fecal calprotectin 539.0 µg/g, interquartile range [IQR] 904.8), and satiety (plasma polypeptide YY 62.6 pmol/l, IQR 110.3) confirm gastrointestinal dysfunction. By day 28, no differences are observable in proportion achieving weight gain >5 g/kg/day (87%, 92%, 86%; p > 0.05), mortality (16%, 30%, 17%; p > 0.05), or edema resolution (83%, 54%, 91%; p > 0.05) among CpF, InF, and ConF. Decreased fecal bacterial richness from day 1 (abundance-based coverage estimator [ACE] 53.2) to day 7 (ACE 40.8) is observed only in ConF (p = 0.025). Bifidobacterium relative abundance increases from day 7 (5.8% ± 8.6%) to day 28 (10.9% ± 8.7%) in CpF (corrected p = 1.000). Legume-enriched feeds support aspects of gut function and the microbiome. Trial registration PACTR201805003381361.

Abietic acid ameliorates psoriasis-like inflammation and modulates gut microbiota in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Abietic acid (AA), an antibacterial terpenoid, was initially isolated from rosin which has been used as a traditional Chinese medicine to treat psoriasis. In our previous works, we found that water-processed rosin (WPR) can alleviate imiquimod (IMQ)-induced psoriasis-like inflammation in mice. However, the efficacy of AA, the main component of WPR, against psoriasis remains unclear. MATERIALS AND METHODS: In this study, we confirmed the anti-psoriasis efficacy of AA (40 mg/kg daily for 7 days) in IMQ-induced psoriasis-like inflammation BALB/c mouse model by the psoriasis area severity index (PASI), flow cytometry, ELISA, histopathological and immunohistochemical analysis. Furthermore, we detected the relative abundance of gut microbe using high-throughput 16S rRNA gene sequencing to validate whether AA modulate gut microbe. RESULT: Oral administration of AA ameliorates IMQ-induced psoriasis-like skin inflammation through reducing PASI scores, regulating the balance of Th17/Treg cells in the mouse spleen, and downregulating the level of serum cytokines such as TNF-α, IL-17A, TGF-1ß, and IL-23. 16S rRNA gene sequencing revealed that the relative abundance of gut bacteria related to inflammation, such as, Anaerotruncus and Christensenella at genus level were decreased, while Kurthia, Citrobacter, and Klebsiella at genus level were increased in AA group mice. Additionally, the correlation analysis illustrated that the key microbiota had a close relationship with the psoriasis-like inflammation related indexes. CONCLUSION: AA might exert the anti-psoriasis effect via inhibiting Th17-related immune responses, hinting that it might be a candidate for treating psoriasis. Meanwhile, the alteration of intestinal microbiota by AA treatment is another possible explanation for the amelioration of imiquimod-induced psoriasis-like inflammation.

Anti-NAFLD effect of defatted walnut powder extract in high fat diet-induced C57BL/6 mice by modulating the gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut kernel has the actions of removing meteorism, dissipating stagnation and removing blood stasis and is used after being defatted in TCM. Defatted walnut powder extract (DWPE) has the abilities of anti-oxidation and lowering lipid levels in vivo. However, the effects and the potential mechanisms of DWPE on NAFLD have not been explored. AIM OF THE STUDY: The study were to investigate the anti-NAFLD effect of DWPE in high fat diet-induced C57BL/6 mice and demonstrate that whether DWPE developed the effect on anti-NAFLD by remodeling the compositions and abundances of gut microbiota. MATERIALS AND METHODS: The inhibitory effect of DWPE on the development of NAFLD was conducted on C57BL/6 mice with a high fat diet and the regulation effect of DWPE on gut microbiota was verified on pseudo-sterile mice with treatment of broad spectrum antibiotics. RESULTS: The results showed that the oral administration of DWPE remarkably alleviated hepatic lipid accumulation by decreasing the levels of TG, TC, LDL, MDA and increasing HDL. Meanwhile, the expressions of NF-κB and MAPKs family proteins were reduced by DWPE compared with HFD group. Otherwise, the efficacy of anti-NAFLD of DWPE was significantly decreased after treatment of antibiotics, which indicated the key role of gut microbiota in the therapeutic process. Furthermore, sequencing of 16S rRNA gene revealed that DWPE could revert the decreased relative abundance of gut microbiota caused by the long term of a high fat diet. And the disordered microflora was remodeled by DWPE including the reduction of Erysipelotrichia, Firmicutes and Actinobacteria as well as the increment of Bacteroidetes, Clostridiales, Bacteroidales S24-7, Prevotellaceae and Bacteroides. CONCLUSION: Taken together, DWPE had a preventing effect on NAFLD, which might be associated with the regulation of gut microbiota.

Alterations in the Intestinal Morphology, Gut Microbiota, and Trace Mineral Status Following Intra-Amniotic Administration (Gallus gallus) of Teff (Eragrostis tef) Seed Extracts.

The consumption of teff (Eragrostis tef), a gluten-free cereal grain, has increased due to its dense nutrient composition including complex carbohydrates, unsaturated fatty acids, trace minerals (especially Fe), and phytochemicals. This study utilized the clinically-validated Gallus gallus intra amniotic feeding model to assess the effects of intra-amniotic administration of teff extracts versus controls using seven groups: (1) non-injected; (2) 18Ω H2O injected; (3) 5% inulin; (4) teff extract 1%; (5) teff extract 2.5%; (6) teff extract 5%; and (7) teff extract 7.5%. The treatment groups were compared to each other and to controls. Our data demonstrated a significant improvement in hepatic iron (Fe) and zinc (Zn) concentration and LA:DGLA ratio without concomitant serum concentration changes, up-regulation of various Fe and Zn brush border membrane proteins, and beneficial morphological changes to duodenal villi and goblet cells. No significant taxonomic alterations were observed using 16S rRNA sequencing of the cecal microbiota. Several important bacterial metabolic pathways were differentially enriched in the teff group, likely due to teff's high relative fiber concentration, demonstrating an important bacterial-host interaction that contributed to improvements in the physiological status of Fe and Zn. Therefore, teff appeared to represent a promising staple food crop and should be further evaluated.

Dietary quinoa (Chenopodium quinoa Willd.) polysaccharides ameliorate high-fat diet-induced hyperlipidemia and modulate gut microbiota.

As the high nutritional and functional values of quinoa acknowledged, the increasing researches focus on the bioactivities and related mechanisms of its abundant carbohydrates. Herein, the beneficial effects of the soluble polysaccharide fraction from quinoa was investigated to lower the serum lipid of rats treated by high-fat diet (HFD) and call the disordered gut microbiota back. The polysaccharide faction was firstly extracted by ultrasonic-assisted extraction technology (yield of 9.65%) and characterized of the monosaccharide composition with glucose and arabinose (1.17:1, molar ratio). And then, the oral administration of quinoa polysaccharide of 300 mg·kg-1·day-1 and 600 mg·kg-1·day-1 for 8 weeks remarkably alleviated dyslipidemia by decreasing the levels of serum total triglyceride (TG), low density lipoprotein cholesterol (LDL-C), malondialdehyde (MDA), total glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) in rats fed with HFD, as well as the reduced hepatic lipid accumulation. Meanwhile, the relative abundance of gut microbiota could be disordered by the long term of HFD. Nevertheless, dietary supplementation of quinoa polysaccharide could enhance species richness and regulate the gut microbiota community structure, reducing the ratio of Firmicutes and Bacteroides, the relative abundance of Proteobacteria. Meanwhile, Sequencing of 16S rRNA gene revealed that intake of quinoa polysaccharide decreased the relative abundances of Desulfovibrio and Allobaculum, which were positively correlated with serum lipid profiles and beneficial to lessen intestinal inflammation. Taken together, the present study demonstrated that quinoa polysaccharide supplementation could ameliorate the hyperlipidemia induced by HFD in association with modulating gut microbiota in a positive way.

Single-molecule correlated chemical probing reveals large-scale structural communication in the ribosome and the mechanism of the antibiotic spectinomycin in living cells.

The ribosome moves between distinct structural states and is organized into multiple functional domains. Here, we examined hundreds of occurrences of pairwise through-space communication between nucleotides in the ribosome small subunit RNA using RNA interaction groups analyzed by mutational profiling (RING-MaP) single-molecule correlated chemical probing in bacterial cells. RING-MaP revealed four structural communities in the small subunit RNA, each distinct from the organization defined by the RNA secondary structure. The head domain contains 2 structural communities: the outer-head contains the pivot for head swiveling, and an inner-head community is structurally integrated with helix 44 and spans the entire ribosome intersubunit interface. In-cell binding by the antibiotic spectinomycin (Spc) barely perturbs its local binding pocket as revealed by the per-nucleotide chemical probing signal. In contrast, Spc binding overstabilizes long-range RNA-RNA contacts that extend 95 Å across the ribosome that connect the pivot for head swiveling with the axis of intersubunit rotation. The two major motions of the small subunit-head swiveling and intersubunit rotation-are thus coordinated via long-range RNA structural communication, which is specifically modulated by Spc. Single-molecule correlated chemical probing reveals trans-domain structural communication and rationalizes the profound functional effects of binding by a low-molecular-mass antibiotic to the megadalton ribosome.

Ceftriaxone and Cefotaxime Have Similar Effects on the Intestinal Microbiota in Human Volunteers Treated by Standard-Dose Regimens.

Ceftriaxone has a higher biliary elimination than cefotaxime (40% versus 10%), which may result in a more pronounced impact on the intestinal microbiota. We performed a monocenter, randomized open-label clinical trial in 22 healthy volunteers treated by intravenous ceftriaxone (1 g/24 h) or cefotaxime (1 g/8 h) for 3 days. We collected fecal samples for phenotypic analyses, 16S rRNA gene profiling, and measurement of the antibiotic concentration and compared the groups for the evolution of microbial counts and indices of bacterial diversity over time. Plasma samples were drawn at day 3 for pharmacokinetic analysis. The emergence of 3rd-generation-cephalosporin-resistant Gram-negative enteric bacilli (Enterobacterales), Enterococcus spp., or noncommensal microorganisms was not significantly different between the groups. Both antibiotics reduced the counts of total Gram-negative enteric bacilli and decreased the bacterial diversity, but the differences between the groups were not significant. All but one volunteer from each group exhibited undetectable levels of antibiotic in feces. Plasma pharmacokinetic endpoints were not correlated to alteration of the bacterial diversity of the gut. Both antibiotics markedly impacted the intestinal microbiota, but no significant differences were detected when standard clinical doses were administered for 3 days. This might be related to the similar daily amounts of antibiotics excreted through the bile using a clinical regimen. (This study has been registered at ClinicalTrials.gov under identifier NCT02659033.).

Antimicrobial Resistance in Mycoplasma spp.

Mycoplasmas are intrinsically resistant to antimicrobials targeting the cell wall (fosfomycin, glycopeptides, or ß-lactam antibiotics) and to sulfonamides, first-generation quinolones, trimethoprim, polymixins, and rifampicin. The antibiotics most frequently used to control mycoplasmal infections in animals are macrolides and tetracyclines. Lincosamides, fluoroquinolones, pleuromutilins, phenicols, and aminoglycosides can also be active. Standardization of methods used for determination of susceptibility levels is difficult since no quality control strains are available and because of species-specific growth requirements. Reduced susceptibility levels or resistances to several families of antimicrobials have been reported in field isolates of pathogenic Mycoplasma species of major veterinary interest: M. gallisepticum and M. synoviae in poultry; M. hyopneumoniae, M. hyorhinis, and M. hyosynoviae in swine; M. bovis in cattle; and M. agalactiae in small ruminants. The highest resistances are observed for macrolides, followed by tetracyclines. Most strains remain susceptible to fluoroquinolones. Pleuromutilins are the most effective antibiotics in vitro. Resistance frequencies vary according to the Mycoplasma species but also according to the countries or groups of animals from which the samples were taken. Point mutations in the target genes of different antimicrobials have been identified in resistant field isolates, in vitro-selected mutants, or strains reisolated after an experimental infection followed by one or several treatments: DNA-gyrase and topoisomerase IV for fluoroquinolones; 23S rRNA for macrolides, lincosamides, pleuromutilins, and amphenicols; 16S rRNAs for tetracyclines and aminoglycosides. Further work should be carried out to determine and harmonize specific breakpoints for animal mycoplasmas so that in vitro information can be used to provide advice on selection of in vivo treatments.

Gut microbiota modulate neurobehavior through changes in brain insulin sensitivity and metabolism.

Obesity and diabetes in humans are associated with increased rates of anxiety and depression. To understand the role of the gut microbiome and brain insulin resistance in these disorders, we evaluated behaviors and insulin action in brain of mice with diet-induced obesity (DIO) with and without antibiotic treatment. We find that DIO mice have behaviors reflective of increased anxiety and depression. This is associated with decreased insulin signaling and increased inflammation in in the nucleus accumbens and amygdala. Treatment with oral metronidazole or vancomycin decreases inflammation, improves insulin signaling in the brain and reduces signs of anxiety and depression. These effects are associated with changes in the levels of tryptophan, GABA, BDNF, amino acids, and multiple acylcarnitines, and are transferable to germ-free mice by fecal transplant. Thus, changes in gut microbiota can control brain insulin signaling and metabolite levels, and this leads to altered neurobehaviors.

Functional metagenomic approach to identify overlooked antibiotic resistance mutations in bacterial rRNA.

Our knowledge as to how bacteria acquire antibiotic resistance is still fragmented, especially for the ribosome-targeting drugs. In this study, with the aim of finding novel mechanisms that render bacteria resistant to the ribosome-targeting antibiotics, we developed a general method to systematically screen for antibiotic resistant 16 S ribosomal RNAs (rRNAs), which are the major target for multiple antibiotics (e.g. spectinomycin, tetracycline, and aminoglycosides), and identify point mutations therein. We used Escherichia coli ∆7, a null mutant of the rrn (ribosomal RNA) operons, as a surrogate host organism to construct a metagenomic library of 16 S rRNA genes from the natural (non-clinical) environment. The library was screened for spectinomycin resistance to obtain four resistant 16 S rRNA genes from non-E. coli bacterial species. Bioinformatic analysis and site-directed mutagenesis identified three novel mutations - U1183C (the first mutation discovered in a region other than helix 34), and C1063U and U1189C in helix 34 - as well as three well-described mutations (C1066U, C1192G, and G1193A). These results strongly suggest that uncharacterized antibiotic resistance mutations still exist, even for traditional antibiotics.

Probiotic potential of Lactobacilli with antagonistic activity against pathogenic strains: An in vitro validation for the production of inhibitory substances.

BACKGROUND: Probiotics, live cells with different beneficiary characteristics, have been extensively studied and explored commercially in many different products in the world. Their benefits to human and animal health have proven in hundreds of scientific studies. Based on rich bibliographic material, Curd is the potential source of probiotic Lactobacilli. METHOD: The aim of the present study was to observe Lactobacilli with probiotic potential activities from different curd samples for isolation, identification and characterization of Lactobacillus species. RESULTS: Among the samples, thirty lactic acid bacterial strains were isolated, sixteen (16/30) best Lactobacillus isolates were selected by preliminary screening as potential probiotic for acid and bile tolerance, further confirmed using 16s rRNA identification. All the selected Lactobacillus isolates were then characterized in vitro for their probiotic characteristics and antimicrobial activities against pathogens and aggregation studies. The results indicated that selected potential probiotic isolates (T2, T4 and T16) were screened and confirmed as Lactobacillus. The isolates produced positive tolerance to excited pH, NaCl and bile salts, also revealed noticeable antimicrobial activities against pathogens. All the Lactobacillus isolates were susceptible to clinical antibiotics used. Besides, T2 isolate was constituted to retain stronger auto and co-aggregation and cell surface hydrophobicity capacity. CONCLUSION: Based on the drawn results, T2, T4 and T16 Lactobacillus isolates were recognised as ideal, potential in vitro antimicrobial probiotic isolates against pathogens and studies are needed further in-vivo assessment and human health benefits in their real-life situations.

16S Ribosomal RNA Gene Sequencing to Evaluate the Effects of 6 Commonly Prescribed Antibiotics.

INTRODUCTION: The rapid antibiotic sensitivity test (RAST) is a novel in-office culture and sensitivity system for endodontic infections. The purpose of this research was to validate the RAST system as a viable, in-office alternative to antibiotic sensitivity testing using turbidity to determine antibiotic sensitivities of endodontic infections. METHODS: Aspirates were taken from the root canals of 9 necrotic human teeth at the initiation of root canal therapy. These samples were cultured in the RAST medium, and antibiotic sensitivity to 6 antibiotics was tested. Further analysis was performed using 16S ribosomal RNA (rRNA) gene sequencing. RESULTS: Thirty-one bacterial phyla were identified as well as 2 phyla of the kingdom Archaea. Augmentin (Dr. Reddy's Laboratories Ltd, Hyderabad, India) and ampicillin performed identically at 24 hours, inhibiting turbidity in 100% of the samples. At 48 hours in anaerobic conditions, Augmentin outperformed ampicillin by 13%. Ciprofloxacin was the least efficacious antibiotic. At 48 hours, only 22% of anaerobic ciprofloxacin cultures affectively inhibited bacterial growth. CONCLUSIONS: The RAST medium is a viable in-office alternative to antibiotic susceptibility testing in an off-site laboratory. It is able to support the growth of a wide variety of microorganisms in both aerobic and anaerobic environments, and, in combination with 16S rRNA gene sequencing, it led to the identification of a new archaebacterial phylum, Crenarchaeota, as part of the endodontic infection microbiome.

Oral treatment with Lactobacillus rhamnosus attenuates behavioural deficits and immune changes in chronic social stress.

BACKGROUND: Stress-related disorders involve systemic alterations, including disruption of the intestinal microbial community. Given the putative connections between the microbiota, immunity, neural function, and behaviour, we investigated the potential for microbe-induced gut-to-brain signalling to modulate the impact of stress on host behaviour and immunoregulation. METHODS: Male C57BL/6 mice treated orally over 28 days with either Lactobacillus rhamnosus (JB-1) ™ or vehicle were subjected to chronic social defeat and assessed for alterations in behaviour and immune cell phenotype. 16S rRNA sequencing and mass spectrometry were employed to analyse the faecal microbial community and metabolite profile. RESULTS: Treatment with JB-1 decreased stress-induced anxiety-like behaviour and prevented deficits in social interaction with conspecifics. However, JB-1 did not alter development of aggressor avoidance following social defeat. Microbial treatment attenuated stress-related activation of dendritic cells while increasing IL-10+ regulatory T cells. Furthermore, JB-1 modulated the effect of stress on faecal metabolites with neuroactive and immunomodulatory properties. Exposure to social defeat altered faecal microbial community composition and reduced species richness and diversity, none of which was prevented by JB-1. Stress-related microbiota disruptions persisted in vehicle-treated mice for 3 weeks following stressor cessation. CONCLUSIONS: These data demonstrate that despite the complexity of the gut microbiota, exposure to a single microbial strain can protect against certain stress-induced behaviours and systemic immune alterations without preventing dysbiosis. This work supports microbe-based interventions for stress-related disorders.

Antimicrobial susceptibility and emerging resistance determinants (blaCTX-M, rmtB, fosA3) in clinical isolates from urinary tract infections in the Bolivian Chaco.

BACKGROUND: Bolivia is among the lowest-resourced South American countries, with very few data available on antibiotic resistance in bacterial pathogens. The phenotypic and molecular characterization of bacterial isolates responsible for urinary tract infections (UTIs) in the Bolivian Chaco are reported here. METHODS: All clinical isolates from UTIs collected in the Hospital Basico Villa Montes between June 2010 and January 2014 were analyzed (N=213). Characterization included susceptibility testing, extended-spectrum beta-lactamase (ESBL) detection, identification of relevant resistance determinants (e.g., CTX-M-type ESBLs, 16S rRNA methyltransferases, glutathione S-transferases), and genotyping of CTX-M producers. RESULTS: Very high resistance rates were observed. Overall, the lowest susceptibility was observed for trimethoprim-sulphamethoxazole, tetracycline, nalidixic acid, amoxicillin-clavulanic acid, ciprofloxacin, and gentamicin. Of E. coli and K. pneumoniae, 11.6% were ESBL producers. Resistance to nitrofurantoin, amikacin, and fosfomycin remained low, and susceptibility to carbapenems was fully preserved. CTX-M-15 was the dominant CTX-M variant. Four E. coli ST131 (two being H30-Rx) were identified. Of note, isolates harbouring rmtB and fosA3 were detected. CONCLUSIONS: Bolivia is not an exception to the very high resistance burden affecting many South American countries. Optimization of alternative approaches to monitor local antibiotic resistance trends in resource-limited settings is strongly encouraged to support the implementation of effective empiric treatment guidelines.

Chemotherapy-driven dysbiosis in the intestinal microbiome.

BACKGROUND: Chemotherapy is commonly used as myeloablative conditioning treatment to prepare patients for haematopoietic stem cell transplantation (HSCT). Chemotherapy leads to several side effects, with gastrointestinal (GI) mucositis being one of the most frequent. Current models of GI mucositis pathophysiology are generally silent on the role of the intestinal microbiome. AIM: To identify functional mechanisms by which the intestinal microbiome may play a key role in the pathophysiology of GI mucositis, we applied high-throughput DNA-sequencing analysis to identify microbes and microbial functions that are modulated following chemotherapy. METHODS: We amplified and sequenced 16S rRNA genes from faecal samples before and after chemotherapy in 28 patients with non-Hodgkin's lymphoma who received the same myeloablative conditioning regimen and no other concomitant therapy such as antibiotics. RESULTS: We found that faecal samples collected after chemotherapy exhibited significant decreases in abundances of Firmicutes (P = 0.0002) and Actinobacteria (P = 0.002) and significant increases in abundances of Proteobacteria (P = 0.0002) compared to samples collected before chemotherapy. Following chemotherapy, patients had reduced capacity for nucleotide metabolism (P = 0.0001), energy metabolism (P = 0.001), metabolism of cofactors and vitamins (P = 0.006), and increased capacity for glycan metabolism (P = 0.0002), signal transduction (P = 0.0002) and xenobiotics biodegradation (P = 0.002). CONCLUSIONS: Our study identifies a severe compositional and functional imbalance in the gut microbial community associated with chemotherapy-induced GI mucositis. The functional pathways implicated in our analysis suggest potential directions for the development of intestinal microbiome-targeted interventions in cancer patients.

Analysis of rpsL and rrs genes mutations related to streptomycin resistance in Mdr and Xdr clinical isolates of Mycobacterium tuberculosis.

INTRODUCTION: Streptomycin is a bactericidal and aminoglycoside antibiotic. It is one of the most effective drugs for treatment of multi-drug Tuberculosis disease. Incidence of resistance is increasingly reported. Its action mechanism is by inhibition of binding aminoacyl tRNA to position "A" in elongation phase, which finally it causes to stop bacterial protein synthesis. In this study, resistance rapid investigation to streptomycin was conducted in clinical strains of Mycobacterium tuberculosis. MATERIALS AND METHODS: In this study, among 105 strains of phlegm-positive and culture-positive Mycobacterium tuberculosis, 45 strains of resistant and sensitive to streptomycin were selected for possible mutations examination in genes rrs and rpsL. Specific primers that used for PCR were named rpsL 1, rpsL 2 and rrsR, Frrs. PCR products were sequenced. RESULT: PCR Products represents 504 bp band for gene rpsL and 1027 bp for gene rrs that shows proper selection of primers and determining an amplification appropriate program. From 26 resistant strains to streptomycin 26 strain have mutation in rpsL gene and 1 strain have alteration in rrs gene. In this study 19 strains were sensitive to streptomycin that have no mutation in these gene. CONCLUSIONS: Streptomycin resistance is mainly related to mutation at codons 43 and 88 "rpsL" gene and to a lesser extent "rrs" that are the greatest cause of drug resistance to streptomycin.

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

Singly modified amikacin and tobramycin derivatives show increased rRNA A-site binding and higher potency against resistant bacteria.

Semisynthetic derivatives of the clinically useful aminoglycosides tobramycin and amikacin were prepared by selectively modifying their 6'' positions with a variety of hydrogen bond donors and acceptors. Their binding to the rRNA A-site was probed using an in vitro FRET-based assay, and their antibacterial activities against several resistant strains (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, MRSA) were quantified by determining minimum inhibitory concentrations (MICs). The most potent derivatives were evaluated for their eukaryotic cytotoxicity. Most analogues displayed higher affinity for the bacterial A-site than the parent compounds. Although most tobramycin analogues exhibited no improvement in antibacterial activity, several amikacin analogues showed potent and broad-spectrum antibacterial activity against resistant bacteria. Derivatives tested for eukaryotic cytotoxicity exhibited minimal toxicity, similar to the parent compounds.

Commonly prescribed ß-lactam antibiotics induce C. trachomatis persistence/stress in culture at physiologically relevant concentrations.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

Mitochondrial translational inhibitors in the pharmacopeia.

The vast majority of energy necessary for cellular function is produced in the mitochondria by the phosphorylation of ADP to ATP. Other critical mitochondrial functions include apoptosis and free-radical production. Chemical agents, including those found in the modern pharmacopeia, may impair mitochondrial function by a number of mechanisms. The mitochondria are vulnerable to environmental injury because of their complex physical structure, electrochemical properties of the inner mitochondrial membrane (IMM), dual genetic control from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) and inherent properties of the translational and transcriptional machinery. Mitochondria have evolved from alpha-proteobacteria and the residual structural similarity to bacterial translational machinery has left the mtDNA genes vulnerable to inhibition by commonly used translation-targeted antibiotics. Many of these medications cause adverse effects in otherwise healthy people, but there are also examples where particular gene mutations may predispose to increased drug toxicity. It is hoped that preclinical pharmacogenetic and functional studies of mitochondrial toxicity, along with personalized genomic medicine, will improve both our understanding of the spectrum of disease caused by inhibition of mitochondrial translation and improve the safe and effective use of antibiotics that inhibit bacterial and human mitochondrial translation. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.