Antibiotics

11 pages, 1251 KiB

Open AccessArticle

Geographic Variation in Antibiotic Consumption—Is It Due to Doctors’ Prescribing or Patients’ Consulting?

by Marte Meyer Walle-Hansen and Sigurd Høye

Antibiotics 2018, 7(1), 26; https://doi.org/10.3390/antibiotics7010026 - 20 Mar 2018

Cited by 7 | Viewed by 7028

Antibiotic consumption varies greatly between Norwegian municipalities. We examine whether this variation is associated with inhabitants’ consultation rates or general practitioners’ (GP) prescription rates. Our study comprises consultations and antibiotic prescriptions for respiratory tract infections (RTIs) in general practice in all Norwegian municipalities [...] Read more.

Antibiotic consumption varies greatly between Norwegian municipalities. We examine whether this variation is associated with inhabitants’ consultation rates or general practitioners’ (GP) prescription rates. Our study comprises consultations and antibiotic prescriptions for respiratory tract infections (RTIs) in general practice in all Norwegian municipalities with over 5000 inhabitants in 2014. Data was collected from The Norwegian Prescription Database, The Directorate of Health’s system for control and payment of health reimbursements registry and Norway Statistics. Consultation rates and prescription rates were categorised in age- and gender specific quintiles and the effect on antibiotic consumption was analysed using a Poisson regression model. We found that inhabitants with RTIs received 42% more prescriptions if they belonged to a municipality with high consultation rates compared to low consultation rates [incidence rate ratio (IRR) 1.42 (95% CI 1.41–1.44)] and 48% more prescriptions if they belonged to a municipality with high prescription rates versus low prescription rates [IRR 1.48 (95% KI 1.47–1.50)]. Our results demonstrate that inhabitants’ consultation rates and GPs’ prescription rates have about equal impact on the number of RTI antibiotics prescribed at municipality level. These findings highlight the importance of interventions targeting patients as well as doctors in efforts to reduce unnecessary antibiotic consumption. Full article

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13 pages, 2658 KiB

Open AccessReview

Novel Aspects of Polynucleotide Phosphorylase Function in Streptomyces

by George H. Jones

Antibiotics 2018, 7(1), 25; https://doi.org/10.3390/antibiotics7010025 - 18 Mar 2018

Cited by 6 | Viewed by 4671

Abstract

Polynucleotide phosphorylase (PNPase) is a 3′–5′-exoribnuclease that is found in most bacteria and in some eukaryotic organelles. The enzyme plays a key role in RNA decay in these systems. PNPase structure and function have been studied extensively in Escherichia coli, but there [...] Read more.

Polynucleotide phosphorylase (PNPase) is a 3′–5′-exoribnuclease that is found in most bacteria and in some eukaryotic organelles. The enzyme plays a key role in RNA decay in these systems. PNPase structure and function have been studied extensively in Escherichia coli, but there are several important aspects of PNPase function in Streptomyces that differ from what is observed in E. coli and other bacterial genera. This review highlights several of those differences: (1) the organization and expression of the PNPase gene in Streptomyces; (2) the possible function of PNPase as an RNA 3′-polyribonucleotide polymerase in Streptomyces; (3) the function of PNPase as both an exoribonuclease and as an RNA 3′-polyribonucleotide polymerase in Streptomyces; (4) the function of (p)ppGpp as a PNPase effector in Streptomyces. The review concludes with a consideration of a number of unanswered questions regarding the function of Streptomyces PNPase, which can be examined experimentally. Full article

(This article belongs to the Special Issue Actinomycetes: The Antibiotics Producers)

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Figure 1
Schematic representation of the Streptomyces coelicolor rpsO–pnp operon. PrpsOA, B and PpnpA, B represent the upstream and intergenic promoters found in S. coelicolor, respectively. The ball-and-stick structures immediately following rpsO and pnp represent rho-independent transcription terminators. The ball-and-stick structure just upstream of pnp represents the intergenic hairpin which is cleaved by RNase III. The diagram is not drawn to scale. Full article ">Figure 2
(A) Growth of the S. coelicolor strains containing promoter probe constructs. Growth was measured as the increase in optical density at 450 nm. The arrows in the figure indicate the onset of the production of two of the secondary metabolites synthesized by S. coelicolor, undecylprodigiosin (red) and actinorhodin (act). (B) Catechol dioxygenase (CATO2ase) activity of mycelial extracts of S. coelicolor derivatives containing the putative rpsOA and rpsOB promoters, cloned in the promoter probe vector pIPP2 [<a href="#B35-antibiotics-07-00025" class="html-bibr">35</a>]. Mycelium was harvested at the indicated times, disrupted by sonication, and following centrifugation, supernatants were assayed for catechol dioxygenase, as described previously [<a href="#B31-antibiotics-07-00025" class="html-bibr">31</a>,<a href="#B35-antibiotics-07-00025" class="html-bibr">35</a>]. The catechol dioxygenase gene is the reporter in the promoter probe vector [<a href="#B35-antibiotics-07-00025" class="html-bibr">35</a>]. (C) CATO2ase activities of extracts of strains containing the putative pnpA and pnpB promoters. The results shown are the averages of duplicate assays from two independent experiments ± SEM. This figure is reprinted from Gene, 536, Patricia Bralley, Marcha L. Gatewood, George H. Jones, Transcription of the rpsO–pnp operon of Streptomyces coelicolor involves four temporally regulated, stress responsive promoters. 177–185, Copyright (2014), with permission from Elsevier [<a href="#B31-antibiotics-07-00025" class="html-bibr">31</a>]. Full article ">Figure 3
Cold shock responses of S. coelicolor. Derivatives containing the rpsO–pnp promoter probe constructs were grown and 30 °C, and half of each culture was then shifted to 10 °C. Mycelium was harvested at the indicated times, disrupted by sonication, and following centrifugation, supernatants were assayed for promoter activity, as described [<a href="#B31-antibiotics-07-00025" class="html-bibr">31</a>,<a href="#B35-antibiotics-07-00025" class="html-bibr">35</a>]. Panel C shows the results of PNPase polymerization assays. In Panels A and B, PNPase promoter activities are expressed relative to the activity measured at 30 °C at zero time, immediately before the shift to 10 °C. The results shown are the averages of duplicate assays from two independent experiments ± SEM. In the first experiment, PNPase levels were measured in S. coelicolor containing PrpsOA and in the second, PNPase levels were measured in the derivative containing PpnpB. This figure is reprinted from Gene, 536, Patricia Bralley, Marcha L. Gatewood, George H. Jones, Transcription of the rpsO-pnp operon of Streptomyces coelicolor involves four temporally regulated, stress responsive promoters. 177–185, Copyright (2014), with permission from Elsevier [<a href="#B31-antibiotics-07-00025" class="html-bibr">31</a>]. Full article ">Figure 4
Effects of nucleoside diphosphates on the phosphorolysis of the 5650 transcript. Phosphorolysis reactions were performed as described in [<a href="#B47-antibiotics-07-00025" class="html-bibr">47</a>], and reaction products were separated by gel electrophoresis. The top panel shows the results obtained with S. coelicolor PNPase and the bottom panel results using E. coli PNPase. Reactions were conducted in the presence of increasing concentrations of a mixture of ADP, CDP, UDP, and GDP (nucleoside diphosphates (NDPs)) as indicated. RP3 is the 5650 transcript, and RP4 represents the product obtained by complete digestion of the intergenic hairpin in RP3 by PNPase. Note that as PNPase is highly processive [<a href="#B48-antibiotics-07-00025" class="html-bibr">48</a>], no intermediates with mobilities between those of RP3 and RP4 were observed. Copyright © American Society for Microbiology (J. Bacteriol. 190, 2008, 98–106, DOI:10.1128/JB.00327-07) [<a href="#B47-antibiotics-07-00025" class="html-bibr">47</a>]. Full article ">Figure 5
Model for the effects of NDPs on the activity S. coelicolor PNPase. The model posits that S. coelicolor PNPase (PacMan symbol) is able to phosphorolyze 5650 and other structured substrates to a limited extent in the absence of NDPs, as indicated by the dashed X. In the presence of NDPs, PNPase synthesizes unstructured 3′-tails in vivo, and these tails then provide an anchor for the enzyme, thus facilitating the digestion of structured substrates. Copyright © American Society for Microbiology (J. Bacteriol. 195, 2013, 5151–5159, DOI:10.1128/JB.00936-13) [<a href="#B49-antibiotics-07-00025" class="html-bibr">49</a>]. Full article ">Figure 6
Effects of (p)ppGpp on the activity of PNPase. Polymerization and phosphorolysis reactions were performed in the absence and presence of guanosine tetraphosphate (ppGpp) or guanosine pentaphosphate (pppGpp), using purified PNPase from S. coelicolor and E. coli [<a href="#B59-antibiotics-07-00025" class="html-bibr">59</a>]. Results are expressed relative to the activities measured in the absence of (p)ppGpp, set arbitrarily to 100%. Full article ">

9 pages, 923 KiB

Open AccessArticle

Parallel Colorimetric Quantification of Choline and Phosphocholine as a Method for Studying Choline Kinase Activity in Complex Mixtures

by Tahl Zimmerman and Salam A. Ibrahim

Antibiotics 2018, 7(1), 24; https://doi.org/10.3390/antibiotics7010024 - 17 Mar 2018

Cited by 8 | Viewed by 4977

Abstract

Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in [...] Read more.

Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in bacterial cell growth and division is not well understood. Phosphocholine production by Chok and its attenuation by inhibitors in the context of complex samples such as cell extracts can currently be quantified by several methods. These include choline depletion measurements, radioactive methods, mass-spectrometry, and nuclear magnetic resonance. The first does not measure phosphocholine directly, the second requires elaborate safety procedures, and the third and fourth require significant capital investments and technical expertise. For these reasons, a less expensive, higher throughput, more easily accessible assay is needed to facilitate further study in Gram-positive Choks. Here, we present the development of a triiodide/activated charcoal/molybdenum blue system for detecting and quantifying choline and phosphocholine in parallel. We demonstrate that this system can reliably quantify changes in choline and phosphocholine concentrations over time in Chok enzymatic assays using cell extracts as the source of the enzyme. This is an easily accessible, convenient, robust, and economical method for studying Chok activity in complex samples. The triiodide/activated charcoal/molybdenum blue system opens new doors into the study choline kinase in Gram-positive pathogens. Full article

(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)

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28 pages, 1054 KiB

Open AccessReview

The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery

by Jon M. Kaguni

Antibiotics 2018, 7(1), 23; https://doi.org/10.3390/antibiotics7010023 - 14 Mar 2018

Cited by 17 | Viewed by 7197

Abstract

DNA replication is an essential process. Although the fundamental strategies to duplicate chromosomes are similar in all free-living organisms, the enzymes of the three domains of life that perform similar functions in DNA replication differ in amino acid sequence and their three-dimensional structures. [...] Read more.

DNA replication is an essential process. Although the fundamental strategies to duplicate chromosomes are similar in all free-living organisms, the enzymes of the three domains of life that perform similar functions in DNA replication differ in amino acid sequence and their three-dimensional structures. Moreover, the respective proteins generally utilize different enzymatic mechanisms. Hence, the replication proteins that are highly conserved among bacterial species are attractive targets to develop novel antibiotics as the compounds are unlikely to demonstrate off-target effects. For those proteins that differ among bacteria, compounds that are species-specific may be found. Escherichia coli has been developed as a model system to study DNA replication, serving as a benchmark for comparison. This review summarizes the functions of individual E. coli proteins, and the compounds that inhibit them. Full article

(This article belongs to the Special Issue Bacterial DNA Replication and Replication Inhibitors)

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11 pages, 491 KiB

Open AccessArticle

Potentially Important Therapeutic Interactions between Antibiotics, and a Specially Engineered Emulsion Drug Vehicle Containing Krill-Oil-Based Phospholipids and Omega-3 Fatty Acids

by David F. Driscoll

Antibiotics 2018, 7(1), 22; https://doi.org/10.3390/antibiotics7010022 - 10 Mar 2018

Cited by 2 | Viewed by 4271

Abstract

The incidence of antimicrobial resistance (AMR) worldwide is increasing as the pipeline for the development of new chemotherapeutic entities is decreasing. Clearly, overexposure to antibiotics, including excessive dosing, is a key factor that fuels AMR. In fact, most of the new antibacterial agents [...] Read more.

The incidence of antimicrobial resistance (AMR) worldwide is increasing as the pipeline for the development of new chemotherapeutic entities is decreasing. Clearly, overexposure to antibiotics, including excessive dosing, is a key factor that fuels AMR. In fact, most of the new antibacterial agents under development are derivatives of existing classes of antibiotics. Novel approaches involving unique antimicrobial combinations, targets, and/or delivery systems are under intense investigation. An innovative combination of active pharmaceutical ingredients (APIs) consisting of antimicrobial drug(s), krill-oil-based phospholipids, and omega-3 fatty acid triglycerides, that may extend the therapeutic viability of currently effective antibiotics, at least until new chemical entities are introduced, is described. Full article

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13 pages, 1759 KiB

Open AccessArticle

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

by Catarina Moreirinha, Nádia Osório, Carla Pereira, Sara Simões, Ivonne Delgadillo and Adelaide Almeida

Antibiotics 2018, 7(1), 21; https://doi.org/10.3390/antibiotics7010021 - 8 Mar 2018

Cited by 6 | Viewed by 4628

Abstract

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. [...] Read more.

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
Spot test results using a phage-resistant mutant of phage AS-A and phage AS-A after first (A) and fifth streak-plating steps (B). Full article ">Figure 2
Scores scatter plot of the IR spectra of phage-resistant colonies A, B and C, along the 5 streak plating steps, and control phage sensitive colonies after 1 (Ct1) and 5 (Ct5) streaking steps. The letters correspond to the different colonies (A is colony A; B is colony B; C is colony C) and the numbers to the streaking-plate days (1 is day 1; 2 is day 2; 3 is day 3; 4 is day 4; 5 is day 5). Full article ">Figure 3
Loadings plot profile of PC1 corresponding to the IR spectra of the phage-resistant colonies A, B and C, along the 5 streak-plating steps, and control phage sensitive colonies. Full article ">Figure 4
(A) SDS PAGE gel of the intracellular proteins of A. salmonicida on first streak-plating. MW, molecular weight marker; Ct, control phage-sensitive A. salmonicida; A is Colony A of the phage-resistant A. salmonicida mutant; B is Colony B of the phage-resistant A. salmonicida mutant; C is Colony C of the phage-resistant A. salmonicida mutant. The marked bands are the ones that showed differential expression between control and clones A, B and C. Band weight is expressed in kilodalton (KDa). (B) Differential expression of the bands, in percentage, comparing Control (phage-sensitive A. salmonicida) with clones A, B and C (phage-resistant A. salmonicida) after 1 streak-plating steps. *** p < 0.001. Full article ">Figure 5
(A) SDS PAGE gel of the intracellular proteins of A. salmonicida on fifth streak plating. MW, molecular weight marker; Ct, control phage-sensitive A. salmonicida; A is Colony A of the phage-resistant A. salmonicida mutant; B is Colony B of the phage-resistant A. salmonicida mutant; C is Colony C of the phage-resistant A. salmonicida mutant. The marked bands are the ones that showed differential expression between control and clones A, B and C. Band weight is expressed in kilodalton (KDa). (B) Differential expression of the bands, in percentage, comparing Control (phage-sensitive A. salmonicida) with clones A, B and C (phage-resistant A. salmonicida) after 5 streak-plating steps. *** p < 0.001. Full article ">

8 pages, 1160 KiB

Open AccessFeature PaperArticle

Biosynthesis of Rishirilide B

by Philipp Schwarzer, Julia Wunsch-Palasis, Andreas Bechthold and Thomas Paululat

Antibiotics 2018, 7(1), 20; https://doi.org/10.3390/antibiotics7010020 - 7 Mar 2018

Cited by 9 | Viewed by 6508

Abstract

Rishirilide B was isolated from Streptomyces rishiriensis and Streptomyces bottropensis on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different ¹³C labelled precursors using the heterologous host Streptomyces albus J1074::cos4 containing [...] Read more.

Rishirilide B was isolated from Streptomyces rishiriensis and Streptomyces bottropensis on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different ¹³C labelled precursors using the heterologous host Streptomyces albus J1074::cos4 containing a cosmid encoding of the gene cluster responsible for rishirilide B production. NMR spectroscopic analysis of labelled compounds demonstrate that the tricyclic backbone of rishirilide B is a polyketide synthesized from nine acetate units. One of the acetate units is decarboxylated to give a methyl group. The origin of the starter unit was determined to be isobutyrate. Full article

(This article belongs to the Special Issue Actinomycetes: The Antibiotics Producers)

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9 pages, 2176 KiB

Open AccessArticle

Genetic Determinants of Tetracycline Resistance in Clinical Streptococcus pneumoniae Serotype 1 Isolates from Niger

by Sani Ousmane, Bouli A. Diallo and Rasmata Ouedraogo

Antibiotics 2018, 7(1), 19; https://doi.org/10.3390/antibiotics7010019 - 6 Mar 2018

Cited by 9 | Viewed by 4991

Abstract

Streptococcus pneumoniae serotype 1 is the first cause of pneumococcal meningitis Niger. To determine the underlying mechanism of resistance to tetracycline in serotype 1 Streptococcus pneumoniae, a collection of 37 isolates recovered from meningitis patients over the period of 2002 to 2009 [...] Read more.

Streptococcus pneumoniae serotype 1 is the first cause of pneumococcal meningitis Niger. To determine the underlying mechanism of resistance to tetracycline in serotype 1 Streptococcus pneumoniae, a collection of 37 isolates recovered from meningitis patients over the period of 2002 to 2009 in Niger were analyzed for drug susceptibility, and whole genome sequencing (WGS) was performed for molecular analyses. MIC level was determined for 31/37 (83.8%) isolates and allowed detection of full resistance (MIC = 8 µg) in 24/31 (77.4%) isolates. No resistance was found to macrolides and quinolones. Sequence-types deduced from WGS were ST217 (54.1%), ST303 (35.1%), ST2206 (5.4%), ST2839 (2.7%) and one undetermined ST (2.7%). All tetracycline resistant isolates carried a Tn5253 like element, which was found to be an association of two smaller transposons of Tn916 and Tn5252 families. No tet(O) and tet(Q) genes were detected. However, a tet(M) like sequence was identified in all Tn5253 positive strains and was found associated to Tn916 composite. Only one isolate was phenotypically resistant to chloramphenicol, wherein a chloramphenicol acetyl transferase (cat) gene sequence homologous to cat_pC194 from the Staphylococcus aureus plasmid pC194 was detected. In conclusion, clinical Streptococcus pneumoniae type 1 isolated during 2002 to 2009 meningitis surveillance in Niger were fully susceptible to macrolides and quinolones but highly resistant to tetracycline (77.4%) through acquisition of a defective Tn5253 like element composed of Tn5252 and Tn916 transposons. Of the 31 tested isolates, only one was exceptionally resistant to chloramphenicol and carried a Tn5253 transposon that contained cat gene sequence. Full article

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Figure 1
Percent antibiotics susceptibility levels of 31 clinical serotype1 Streptococcus pneumoniae recovered from meningitis patients during 2002–2009 meningitis surveillance in Niger. Legend: S = Susceptible; R = Resistant; PENI = Penicillin; ERY= Erythromycin; CLIND = Clindamycin; CHL = Chloramphenicol; TET = Tetracycline; CIP = Ciprofloxacin. Full article ">Figure 2
Annual percent distribution of tetracycline resistant Streptococcus pneumoniae serotype 1 recovered from meningitis cases from 2002 to 2009 in Niger. The figure shows that resistant isolates prevailed over susceptible ones every year except in 2009. No isolate from 2007 was tested. Full article ">Figure 3
Evolutionary diversity of tet(M) genes from clinical serotype 1 Streptococcus pneumoniae recovered from meningitis cases between 2002 and 2009 in Niger. The analysis involved 32 tet(M) sequences and a reference sequence. PN34 (Accession number: AY466395.1). Sequence names were coded as NIG for Niger followed by isolate identity number, underscore and the year of isolation. Taxa fall in two clades each split into 2 subclades. Full article ">Figure 4
Comparative analysis of Tn5253 mobile element (accession N° gi|284803504| to Tn5253 like sequence from genomes of type 1 clinical Streptococcus pneumoniae strains NIG0588_02 representing isolates susceptible to chloramphenicol and strain NIG1144_06 that was chloramphenicol resistant. Full article ">

17 pages, 1222 KiB

Open AccessArticle

Efflux Activity Differentially Modulates the Levels of Isoniazid and Rifampicin Resistance among Multidrug Resistant and Monoresistant Mycobacterium tuberculosis Strains

by Diana Machado, João Perdigão, Isabel Portugal, Marco Pieroni, Pedro A. Silva, Isabel Couto and Miguel Viveiros

Antibiotics 2018, 7(1), 18; https://doi.org/10.3390/antibiotics7010018 - 3 Mar 2018

Cited by 23 | Viewed by 5095

Abstract

With the growing body of knowledge on the contribution of efflux activity to Mycobacterium tuberculosis drug resistance, increased attention has been given to the use of efflux inhibitors as adjuvants of tuberculosis therapy. Here, we investigated how efflux activity modulates the levels of [...] Read more.

With the growing body of knowledge on the contribution of efflux activity to Mycobacterium tuberculosis drug resistance, increased attention has been given to the use of efflux inhibitors as adjuvants of tuberculosis therapy. Here, we investigated how efflux activity modulates the levels of efflux between monoresistant and multi- and extensively drug resistant (M/XDR) M. tuberculosis clinical isolates. The strains were characterized by antibiotic susceptibility testing in the presence/absence of efflux inhibitors, molecular typing, and genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. The results demonstrated that all the M. tuberculosis clinical strains, susceptible or resistant, presented a faster, rapid, and non-specific efflux-mediated short-term response to drugs. The synergism assays demonstrated that the efflux inhibitors were more effective in reducing the resistance levels in the M/XDR strains than in the monoresistant strains. This indicated that M/XDR strains presented a more prolonged response to drugs mediated by efflux compared to the monoresistant strains, but both maintain it as a long-term stress response. This work shows that efflux activity modulates the levels of drug resistance between monoresistant and M/XDR M. tuberculosis clinical strains, allowing the bacteria to survive in the presence of noxious compounds. Full article

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Figure 1
Schematic representation of parameters used for the characterization of the efflux activity of the M. tuberculosis strains. Presented in the Figure are the results obtained for the H37Rv strain. (A) SpanEtBr corresponds to the difference between the ethidium bromide fluorescence values at t0 of the highest concentration tested (Yhc) and the fluorescence value at t0 of the equilibrium concentration of ethidium bromide (Yeqc); (B) RFF (relative final fluorescence) is a measure of how effective a compound is on the inhibition of ethidium bromide efflux (at a given concentration) by comparison of the final fluorescence at the last time point (60 min) of the treated cells with the cells in presence of ethidium bromide only [<a href="#B19-antibiotics-07-00018" class="html-bibr">19</a>,<a href="#B32-antibiotics-07-00018" class="html-bibr">32</a>]; (C) efflux rate constant, or K value, and the tefflux50% that corresponds to the time required for the cells to extrude half of the preloaded dye; the efflux of ethidium bromide is initiated at t0 by the addition of 0.4% glucose and is 50% complete at tefflux50%. Full article ">Figure 2
Accumulation and efflux of ethidium bromide of the M. tuberculosis strains. (A) Accumulation of ethidium bromide in the presence of efflux inhibitors. In these cases, the strains were loaded with 0.25 µg/mL of ethidium bromide in the presence of verapamil or thioridazine at ½ MIC; (B) Efflux of ethidium bromide. Strains were loaded with ethidium bromide at 0.25 µg/mL; efflux took place in the presence of glucose which was inhibited by verapamil at ½ MIC. Full article ">

25 pages, 902 KiB

Open AccessReview

Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care

by Michael J. Love, Dinesh Bhandari, Renwick C. J. Dobson and Craig Billington

Antibiotics 2018, 7(1), 17; https://doi.org/10.3390/antibiotics7010017 - 27 Feb 2018

Cited by 121 | Viewed by 12730

Abstract

There is growing concern about the emergence of bacterial strains showing resistance to all classes of antibiotics commonly used in human medicine. Despite the broad range of available antibiotics, bacterial resistance has been identified for every antimicrobial drug developed to date. Alarmingly, there [...] Read more.

There is growing concern about the emergence of bacterial strains showing resistance to all classes of antibiotics commonly used in human medicine. Despite the broad range of available antibiotics, bacterial resistance has been identified for every antimicrobial drug developed to date. Alarmingly, there is also an increasing prevalence of multidrug-resistant bacterial strains, rendering some patients effectively untreatable. Therefore, there is an urgent need to develop alternatives to conventional antibiotics for use in the treatment of both humans and food-producing animals. Bacteriophage-encoded lytic enzymes (endolysins), which degrade the cell wall of the bacterial host to release progeny virions, are potential alternatives to antibiotics. Preliminary studies show that endolysins can disrupt the cell wall when applied exogenously, though this has so far proven more effective in Gram-positive bacteria compared with Gram-negative bacteria. Their potential for development is furthered by the prospect of bioengineering, and aided by the modular domain structure of many endolysins, which separates the binding and catalytic activities into distinct subunits. These subunits can be rearranged to create novel, chimeric enzymes with optimized functionality. Furthermore, there is evidence that the development of resistance to these enzymes may be more difficult compared with conventional antibiotics due to their targeting of highly conserved bonds. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
Life cycle of a virulent tailed phage (not to scale). (1) The phage collides with the bacterial cell; (2) the phage binds to cell receptors; (3) the phage is irreversibly bound and injects nucleic acid into the cell via the tail tube, where it is transcribed and translated; (4) many progeny phages are produced within intact cells; (5) endolysins degrade the host bacterial cell wall, which loses its structural integrity and ruptures due to the osmotic pressure, releasing the progeny phages. Full article ">Figure 2
Diagram of the typical cell wall and peptidoglycan structure of bacteria, including the endolysin cleavage sites. The peptidoglycan is composed of repeating sugar units, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked via an interpeptide bridge between the meso-diaminopimelic acid (m-DAP) and d-alanine (d-Ala) residues of adjacent tetrapeptide chains. The chains also contain L-alanine (l-Ala) and D-glutamic acid (d-Glu). Gram-negative bacteria contain an outer membrane (OM) structure not present in Gram-positive bacteria. Both contain an inner membrane (IM) structure. The cleaved bonds and major classifications of endolysin are indicated: (1) N-acetylmuramoyl-l-alanine amidase; (2–4) various endopeptidases; (5) N-acetyl-β-d-glucosaminidase; (6) N-acetyl-β-d-muramidase (lysozyme). Full article ">

11 pages, 2604 KiB

Open AccessArticle

Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish

by Tuan Son Le, Thi Hien Nguyen, Hong Phuong Vo, Van Cuong Doan, Hong Loc Nguyen, Minh Trung Tran, Trong Tuan Tran, Paul C. Southgate and D. İpek Kurtböke

Antibiotics 2018, 7(1), 16; https://doi.org/10.3390/antibiotics7010016 - 25 Feb 2018

Cited by 69 | Viewed by 9425

Abstract

To determine the effectivity of bacteriophages in controlling the mass mortality of striped catfish (Pangasianodon hypophthalmus) due to infections caused by Aeromonas spp. in Vietnamese fish farms, bacteriophages against pathogenic Aeromonas hydrophila were isolated. A. hydrophila-phage 2 and A. hydrophila [...] Read more.

To determine the effectivity of bacteriophages in controlling the mass mortality of striped catfish (Pangasianodon hypophthalmus) due to infections caused by Aeromonas spp. in Vietnamese fish farms, bacteriophages against pathogenic Aeromonas hydrophila were isolated. A. hydrophila-phage 2 and A. hydrophila-phage 5 were successfully isolated from water samples from the Saigon River of Ho Chi Minh City, Vietnam. These phages, belonging to the Myoviridae family, were found to have broad activity spectra, even against the tested multiple-antibiotic-resistant Aeromonas isolates. The latent periods and burst size of phage 2 were 10 min and 213 PFU per infected host cell, respectively. The bacteriophages proved to be effective in inhibiting the growth of the Aeromonas spp. under laboratory conditions. Phage treatments applied to the pathogenic strains during infestation of catfish resulted in a significant improvement in the survival rates of the tested fishes, with up to 100% survival with MOI 100, compared to 18.3% survival observed in control experiments. These findings illustrate the potential for using phages as an effective bio-treatment method to control Motile Aeromonas Septicemia (MAS) in fish farms. This study provides further evidence towards the use of bacteriophages to effectively control disease in aquaculture operations. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
Plaque formation and microphotograph of A. hydrophila phages. (a,b) Φ2 and (c,d) phage Φ5. Full article ">Figure 2
Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila-phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively. Full article ">Figure 3
(a) Adsorption rate and (b) one-step growth curves of Φ2. Full article ">Figure 3 Cont.
(a) Adsorption rate and (b) one-step growth curves of Φ2. Full article ">Figure 4
Inactivation of A. hydrophila N17 by the phages (a) Φ2 and (b) Φ5 at different MOI (0.01, 0.1 and 1). Full article ">Figure 5
Cumulative mortality rates (%) of striped catfishes obtained in challenging experiments using A. hydrophia N17 and the phage cocktail at the different MOIs (0.01, 0.1, and 1). The ratio of Φ2 to Φ5 in a phage cocktail was 1:1. Full article ">

23 pages, 1810 KiB

Open AccessReview

Bacteriophage Interactions with Marine Pathogenic Vibrios: Implications for Phage Therapy

by Panos G. Kalatzis, Daniel Castillo, Pantelis Katharios and Mathias Middelboe

Antibiotics 2018, 7(1), 15; https://doi.org/10.3390/antibiotics7010015 - 24 Feb 2018

Cited by 73 | Viewed by 11425

Abstract

A global distribution in marine, brackish, and freshwater ecosystems, in combination with high abundances and biomass, make vibrios key players in aquatic environments, as well as important pathogens for humans and marine animals. Incidents of Vibrio-associated diseases (vibriosis) in marine aquaculture are [...] Read more.

A global distribution in marine, brackish, and freshwater ecosystems, in combination with high abundances and biomass, make vibrios key players in aquatic environments, as well as important pathogens for humans and marine animals. Incidents of Vibrio-associated diseases (vibriosis) in marine aquaculture are being increasingly reported on a global scale, due to the fast growth of the industry over the past few decades years. The administration of antibiotics has been the most commonly applied therapy used to control vibriosis outbreaks, giving rise to concerns about development and spreading of antibiotic-resistant bacteria in the environment. Hence, the idea of using lytic bacteriophages as therapeutic agents against bacterial diseases has been revived during the last years. Bacteriophage therapy constitutes a promising alternative not only for treatment, but also for prevention of vibriosis in aquaculture. However, several scientific and technological challenges still need further investigation before reliable, reproducible treatments with commercial potential are available for the aquaculture industry. The potential and the challenges of phage-based alternatives to antibiotic treatment of vibriosis are addressed in this review. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
Massive mortalities caused by vibriosis in different developmental stages. (a,b) cultured European seabass, Dicentrarchus labrax, (c) cultured European seabass, Dicentrarchus labrax fry and (d) cultured gilthead sea bream, Sparus aurata larvae in the hatchery. Full article ">Figure 2
Facilities for live feed production from a commercial fish farm unit. (a) Artemia salina in culture tanks with vigorous aeration, where the native presumptive Vibrio load is regularly estimated between 107 and 108 cells per mL; (b) Brachionus plicatilis culture tanks, where the native presumptive Vibrio load is regularly between 102 and 108 cells per mL [<a href="#B103-antibiotics-07-00015" class="html-bibr">103</a>]. Full article ">Figure 3
Overview of the main phage defense mechanisms in bacteria. Prevention of viral attachment on the bacterial surface can be achieved by mutating or masking the receptors, as well as downregulation of receptor expression, orchestrated by quorum sensing (QS). DNA injection may be successfully averted by superinfection exclusion (Sie) mechanisms. If phage DNA enters the bacterial host, its digestion can be catalyzed by R-M mechanism and CRISPR-Cas arrays systems. Deliberate death of the infected cell (abortive infection) constitutes another strategy against viral predators, where prevention of phage proliferation reduces spreading of the infection to the rest of the population. Full article ">

12 pages, 6193 KiB

Open AccessArticle

Fragment-Based Discovery of Inhibitors of the Bacterial DnaG-SSB Interaction

by Zorik Chilingaryan, Stephen J. Headey, Allen T. Y. Lo, Zhi-Qiang Xu, Gottfried Otting, Nicholas E. Dixon, Martin J. Scanlon and Aaron J. Oakley

Antibiotics 2018, 7(1), 14; https://doi.org/10.3390/antibiotics7010014 - 22 Feb 2018

Cited by 14 | Viewed by 4767

Abstract

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which Escherichia coli DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide [...] Read more.

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which Escherichia coli DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is highly conserved and is known to engage in essential interactions with many proteins in nucleic acid metabolism, including primase. Here, fragment-based screening by saturation-transfer difference nuclear magnetic resonance (STD-NMR) and surface plasmon resonance assays identified inhibitors of the primase/SSB-Ct interaction. Hits were shown to bind to the SSB-Ct-binding site using ¹⁵N–¹H HSQC spectra. STD-NMR was used to demonstrate binding of one hit to other SSB-Ct binding partners, confirming the possibility of simultaneous inhibition of multiple protein/SSB interactions. The fragment molecules represent promising scaffolds on which to build to discover new antibacterial compounds. Full article

(This article belongs to the Special Issue Bacterial DNA Replication and Replication Inhibitors)

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15 pages, 4649 KiB

Open AccessArticle

Efficacy of an Optimised Bacteriophage Cocktail to Clear Clostridium difficile in a Batch Fermentation Model

by Janet Y. Nale, Tamsin A. Redgwell, Andrew Millard and Martha R. J. Clokie

Antibiotics 2018, 7(1), 13; https://doi.org/10.3390/antibiotics7010013 - 13 Feb 2018

Cited by 69 | Viewed by 9730

Abstract

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea. Conventional antibiotics are not universally effective for all ribotypes, and can trigger dysbiosis, resistance and recurrent infection. Thus, novel therapeutics are needed to replace and/or supplement the current antibiotics. Here, we describe [...] Read more.

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea. Conventional antibiotics are not universally effective for all ribotypes, and can trigger dysbiosis, resistance and recurrent infection. Thus, novel therapeutics are needed to replace and/or supplement the current antibiotics. Here, we describe the activity of an optimised 4-phage cocktail to clear cultures of a clinical ribotype 014/020 strain in fermentation vessels spiked with combined fecal slurries from four healthy volunteers. After 5 h, we observed ~6-log reductions in C. difficile abundance in the prophylaxis regimen and complete C. difficile eradication after 24 h following prophylactic or remedial regimens. Viability assays revealed that commensal enterococci, bifidobacteria, lactobacilli, total anaerobes, and enterobacteria were not affected by either regimens, but a ~2-log increase in the enterobacteria, lactobacilli, and total anaerobe abundance was seen in the phage-only-treated vessel compared to other treatments. The impact of the phage treatments on components of the microbiota was further assayed using metagenomic analysis. Together, our data supports the therapeutic application of our optimised phage cocktail to treat CDI. Also, the increase in specific commensals observed in the phage-treated control could prevent further colonisation of C. difficile, and thus provide protection from infection being able to establish. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
Contributory culturable bacterial counts from each of the individual donors and final cumulative counts of each bacterium added to the fermentation vessels. The bacteria present in the fecal sample of each donor were determined by recovery on selective medium for each bacterial grouping, after which, the samples were mixed together in relatively equal amounts and used to prime the fermentation vessels. The data was analysed using GraphPad Prism 7. Error bars are SEMs of three biological replicates. Full article ">Figure 2
Impact of phage treatment on C. difficile and other components of the gut microbiota. The impact of phage treatment was ascertained by recovering the bacteria on selective media for (A) C. difficile; (B) bifidobacteria; (C) enterococci; (D) enterobacteria; (E) lactobacilli; (F) total anaerobes. The bacterial counts of the different treatment vessels and time points are presented. Black lines, vessel 1, untreated slurries; red lines, vessel 2, C. difficile control; green lines, vessel 3, phage-only-treated control; blue lines, vessel 4, prophylaxis regimen, and purple lines, vessel 5, remedial regimen. The data was analysed using GraphPad Prism 7. Error bars are SEMs of 3 biological replicates. Full article ">Figure 3
Analysis of the 10 most abundant taxa from Archea, Bacteria, and Viruses as ascertained by the metagenomics data. Total genomic DNA was extracted at 24 h time point from (A) vessel 1, untreated slurries; (B) vessel 2, C. difficile control; (C) vessel 3, phage-only-treated control; (D) vessel 4, prophylaxis regimen; (E) vessel 5, remedial regimen. The samples were prepared using NexteraXT sample preparation kit and sequenced on MiSeq platform using V2 (2 × 250 bp) chemistry. The resulting fastq files were trimmed with Sickle, and the metagenomes were assembled using megahit. An overview of the 10 most abundant taxa: Phyla (P), Classes (C), order (O), and family (F) are shown for each treatment vessel, as visualised using Pavian. The percent reads mapped to Archaea, Bacteria, and Viruses in the vessels at 24 h are shown in (Fi), (Fii), and (Fiii), respectively. Full article ">

15 pages, 1970 KiB

Open AccessArticle

Diversification of Secondary Metabolite Biosynthetic Gene Clusters Coincides with Lineage Divergence in Streptomyces

by Mallory J. Choudoir, Charles Pepe-Ranney and Daniel H. Buckley

Antibiotics 2018, 7(1), 12; https://doi.org/10.3390/antibiotics7010012 - 13 Feb 2018

Cited by 36 | Viewed by 6492

Abstract

We have identified Streptomyces sister-taxa which share a recent common ancestor and nearly identical small subunit (SSU) rRNA gene sequences, but inhabit distinct geographic ranges demarcated by latitude and have sufficient genomic divergence to represent distinct species. Here, we explore the evolutionary dynamics [...] Read more.

We have identified Streptomyces sister-taxa which share a recent common ancestor and nearly identical small subunit (SSU) rRNA gene sequences, but inhabit distinct geographic ranges demarcated by latitude and have sufficient genomic divergence to represent distinct species. Here, we explore the evolutionary dynamics of secondary metabolite biosynthetic gene clusters (SMGCs) following lineage divergence of these sister-taxa. These sister-taxa strains contained 310 distinct SMGCs belonging to 22 different gene cluster classes. While there was broad conservation of these 22 gene cluster classes among the genomes analyzed, each individual genome harbored a different number of gene clusters within each class. A total of nine SMGCs were conserved across nearly all strains, but the majority (57%) of SMGCs were strain-specific. We show that while each individual genome has a unique combination of SMGCs, this diversity displays lineage-level modularity. Overall, the northern-derived (NDR) clade had more SMGCs than the southern-derived (SDR) clade (40.7 ± 3.9 and 33.8 ± 3.9, mean and S.D., respectively). This difference in SMGC content corresponded with differences in the number of predicted open reading frames (ORFs) per genome (7775 ± 196 and 7093 ± 205, mean and S.D., respectively) such that the ratio of SMGC:ORF did not differ between sister-taxa genomes. We show that changes in SMGC diversity between the sister-taxa were driven primarily by gene acquisition and deletion events, and these changes were associated with an overall change in genome size which accompanied lineage divergence. Full article

(This article belongs to the Special Issue Actinomycetes: The Antibiotics Producers)

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The northern-derived (NDR) and southern-derived (SDR) clades are closely related sister-taxa and yet were isolated from soils of different latitude. The un-rooted tree was constructed from multiple whole genome alignments with maximum likelihood and a GTRGAMMA model of evolution. Scale bar represents nucleotide substitutions per site. Colored branches depict the northern-derived (NDR) and southern-derived (SDR) clades. Strain names reflect the sample site they were isolated from (<a href="#app1-antibiotics-07-00012" class="html-app">Table S1</a>). Genome NBRC 13350 is the publically available type strain Streptomyces griseus subsp. griseus NBRC 13350. Sample locations are shown in the right panel and labeled with the site code. Circles are colored to reflect the geographic distribution of clades. (Figure modified from [<a href="#B29-antibiotics-07-00012" class="html-bibr">29</a>]). Full article ">Figure 2
NDR strains have more secondary metabolite biosynthetic gene clusters (SMGCs) than SDR strains (t-test, p < 0.001). (a). Bars indicate the number of SMGCs identified in each genome and are colored according to clade affiliation, and genome names reflect the site of isolation as identified in <a href="#app1-antibiotics-07-00012" class="html-app">Table S1</a>; (b). Kernal density plot shows the distribution of SMGC length (bp). Full article ">Figure 3
A total of 22 SMGC classes were observed in NDR and SDR genomes by antiSMASH [<a href="#B32-antibiotics-07-00012" class="html-bibr">32</a>]. The tree reflects phylogenetic relationships between Streptomyces sister-taxa genomes and was constructed from multiple whole genome alignments (see <a href="#antibiotics-07-00012-f001" class="html-fig">Figure 1</a>). Scale bar represents nucleotide substitutions per site. Tree branches are colored according to clade affiliation. Bars depict the number of gene clusters belonging to each class for each genome. Colors illustrate gene cluster class as provided by the legend. Asterisks note gene cluster classes that are significantly enriched between clades (t-test and Bonferonni correction for multiple comparisons, p < 0.002). Full article ">Figure 4
The frequency distribution of SMGCs across strains shows that most SMGCs are strain-specific and fewer are species-specific. Results are shown both for NDR and SDR. (a) and for all 24 genomes; (b). Non-redundant orthologous SMGCs were defined using our annotation-independent approach (see Materials and Methods). Full article ">Figure 5
We identified 310 non-redundant distinct SMGCs using our annotation-independent gene clustering approach (see Materials and Methods). Each point represents a unique SMGC from a single genome, and colors correspond to clade affiliation. SMGCs with a similar gene composition are clustered spatially, and cluster membership is depicted with polygons. The same data is presented in a different network diagram in <a href="#app1-antibiotics-07-00012" class="html-app">Figure S1</a>. Full article ">Figure 6
A total of nine core (i.e., conserved in ≥80% of genomes) SMGCs were found in both NDR and SDR. The NDR clade had 11 core SMGCs and the SDR clade had 15 core SMGCs. The tree reflects phylogenetic relationships between Streptomyces sister-taxa genomes and was constructed from multiple whole genome alignments (see <a href="#antibiotics-07-00012-f001" class="html-fig">Figure 1</a>). Scale bar represents nucleotide substitutions per site. Tree branches are colored according to clade affiliation. Core orthologous SMGCs (depicted by colored circles) were determined using the antiSMASH [<a href="#B32-antibiotics-07-00012" class="html-bibr">32</a>] MIBiG annotation pipeline or were defined using our annotation-independent approach (see Materials and Method). Colors correspond to SMGC class (see legend), and natural product annotations are labeled if available. Full article ">Figure 7
Gene content of core SMGCs vary within and between clades as a result of gene acquisition and deletion events. Panels depict the gene content (i.e., genetic architecture) of core SMGCs (i.e., conserved in ≥80% of genomes), the NDR-specific SMGC core, and the SDR-specific SMGC core. Black bars within the panels represent orthologous genes. The tree reflects phylogenetic relationships between Streptomyces sister-taxa genomes and was constructed from multiple whole genome alignments (see <a href="#antibiotics-07-00012-f001" class="html-fig">Figure 1</a>). Scale bar represents nucleotide substitutions per site. Panel colors correspond to SMGC class (see legend). Panels are labeled with the SMGC cluster membership (see <a href="#app1-antibiotics-07-00012" class="html-app">Table S3</a>) defined using our annotation-independent approach (see Materials and Methods). Full article ">

7 pages, 410 KiB

Open AccessArticle

Survey of Nonprescription Medication and Antibiotic Use in Patients with Stevens-Johnson Syndrome, Toxic Epidermal Necrolysis, and Overlap Syndrome

by Katherine J. Sullivan, Meghan N. Jeffres, Robert P. Dellavalle, Robert Valuck and Heather D. Anderson

Antibiotics 2018, 7(1), 11; https://doi.org/10.3390/antibiotics7010011 - 1 Feb 2018

Cited by 3 | Viewed by 6744

Abstract

Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and overlap syndrome (SJS-TEN) are rare, serious skin and mucosa break-down conditions frequently associated with antibiotic use. The role of nonprescription medications alone, or in combination with antibiotics in triggering SJS/TEN, is largely unknown. This study [...] Read more.

Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and overlap syndrome (SJS-TEN) are rare, serious skin and mucosa break-down conditions frequently associated with antibiotic use. The role of nonprescription medications alone, or in combination with antibiotics in triggering SJS/TEN, is largely unknown. This study summarized data collected from patient surveys about nonprescription and antibiotic use prior to a SJS/TEN diagnosis. The survey was administered online to members of the U.S. SJS Foundation who had been diagnosed with SJS/TEN or were the parent of a child who had been diagnosed with SJS/TEN. Respondents were asked about nonprescription medications taken within the year before diagnosis, and the approximate point in time before diagnosis that they had taken them. They were also asked about specific prescription medications, including antibiotics, that they took before diagnosis. An estimated 4500 patients received an invitation to complete the survey. 251 patients completed it, resulting in a response rate of 5.6%. The mean age of respondents was 43 years (SD (standard deviation) = 17.3) and 70% were female. 32.3% of respondents indicated that a prescription antibiotic triggered their reaction. 14.1% indicated a nonprescription medication had triggered their SJS/TEN, and 18.1% said a nonprescription medication may have triggered their SJS/TEN. 85.5% of respondents said they took a nonprescription medication within three months of their SJS/TEN diagnosis. Of those respondents who reported that an antibiotic triggered their SJS/TEN, 35.2% reported taking a nonprescription medication within the three months prior to their diagnosis. This survey captured valuable information about nonprescription and antibiotic use in SJS/TEN patients. It is important for future studies to estimate the impact of antibiotics on SJS/TEN, and account for nonprescription medication use in that relationship. Full article

(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)

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8 pages, 225 KiB

Open AccessArticle

Assessing the Knowledge, Attitudes and Behaviors of Human and Animal Health Students towards Antibiotic Use and Resistance: A Pilot Cross-Sectional Study in the UK

by Oliver James Dyar, Holly Hills, Lara-Turiya Seitz, Alex Perry and Diane Ashiru-Oredope

Antibiotics 2018, 7(1), 10; https://doi.org/10.3390/antibiotics7010010 - 30 Jan 2018

Cited by 60 | Viewed by 10713

Abstract

The Global Action Plan on Antimicrobial Resistance highlights the importance of training all healthcare professionals. No study has assessed patterns of students’ knowledge, attitudes and practices concerning antibiotic use simultaneously across different healthcare course types. We conducted a cross-sectional multi-center survey among UK [...] Read more.

The Global Action Plan on Antimicrobial Resistance highlights the importance of training all healthcare professionals. No study has assessed patterns of students’ knowledge, attitudes and practices concerning antibiotic use simultaneously across different healthcare course types. We conducted a cross-sectional multi-center survey among UK students. The survey was advertised through local survey coordinators at 25 universities. The online survey was accessible from 10th October to 17th November 2016 (before European Antibiotic Awareness Day). A total of 255 students from 25 universities participated, including students on medicine, pharmacy, nursing, physician associate, dentistry and veterinary medicine courses. Antibiotic resistance was considered to be a more important global challenge than climate change, obesity or food security (p < 0.001). Most students (95%) believed that antibiotic resistance will be a problem for their future practice, but fewer (69%) thought that the antibiotics they will prescribe, administer or dispense will contribute to the problem. A fifth of students felt they had sufficient knowledge of antibiotic use for their future work. Our exploratory study suggests that UK human and animal healthcare students are aware of the importance of antibiotic resistance, but many still have certain misconceptions. Campaigns and improved educational efforts applying behavioral insights methodology could address these. Full article

(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)

16 pages, 1965 KiB

Open AccessArticle

Use of a Regression Model to Study Host-Genomic Determinants of Phage Susceptibility in MRSA

by Henrike Zschach, Mette V. Larsen, Henrik Hasman, Henrik Westh, Morten Nielsen, Ryszard Międzybrodzki, Ewa Jończyk-Matysiak, Beata Weber-Dąbrowska and Andrzej Górski

Antibiotics 2018, 7(1), 9; https://doi.org/10.3390/antibiotics7010009 - 29 Jan 2018

Cited by 5 | Viewed by 4627

Abstract

Staphylococcus aureus is a major agent of nosocomial infections. Especially in methicillin-resistant strains, conventional treatment options are limited and expensive, which has fueled a growing interest in phage therapy approaches. We have tested the susceptibility of 207 clinical S. aureus strains to 12 [...] Read more.

Staphylococcus aureus is a major agent of nosocomial infections. Especially in methicillin-resistant strains, conventional treatment options are limited and expensive, which has fueled a growing interest in phage therapy approaches. We have tested the susceptibility of 207 clinical S. aureus strains to 12 (nine monovalent) different therapeutic phage preparations and subsequently employed linear regression models to estimate the influence of individual host gene families on resistance to phages. Specifically, we used a two-step regression model setup with a preselection step based on gene family enrichment. We show that our models are robust and capture the data’s underlying signal by comparing their performance to that of models build on randomized data. In doing so, we have identified 167 gene families that govern phage resistance in our strain set and performed functional analysis on them. This revealed genes of possible prophage or mobile genetic element origin, along with genes involved in restriction-modification and transcription regulators, though the majority were genes of unknown function. This study is a step in the direction of understanding the intricate host-phage relationship in this important pathogen with the outlook to targeted phage therapy applications. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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Figure 1
All-against-all matrix of the genetic distance between the 207 methicillin-resistant Staphylococcus aureus (MRSA) strains used for this study. Distance is calculated as 1-orthoANI and represented as color, where blue corresponds to lower and red corresponds to greater distance. The assignment of strains to partitions is marked on the right margin. Full article ">Figure 2
Abundance of gene families in the 207 strains. The peak depicted in the histogram is slightly higher than the number of housekeeping genes, 1.777, since the bin is wider than 1. Full article ">Figure 3
Stacked histogram of p-value distributions across the five folds for the interaction with phage P4/6409. The density is shown instead of counts to account for fold 1 having a 100 times less p-values compared to the other folds, since it does not include partition 1 and therefore did not need to be subsampled. Left: Real data. Right: Permuted data. Full article ">Figure 4
Heat map of the regression weights for the final model of phage P4/6409. Columns are gene families, rows are cross validation folds. The color indicates the value and direction of each weight, with blue being strongly positive and red being strongly negative. Weights with low values are white. Results were comparable for other phages with the exception of 1N/80, A3/R, and mix MS-1 (see <a href="#antibiotics-07-00009-t002" class="html-table">Table 2</a>). Full article ">Figure 5
Functional annotation categories of the eggNOGs matching to the set of significant genes across all nine phages. Full article ">Figure 6
Histogram depicting the number of phage models where a given gene family was identified significant. Full article ">

25 pages, 1303 KiB

Open AccessArticle

Phage-Bacterial Dynamics with Spatial Structure: Self Organization around Phage Sinks Can Promote Increased Cell Densities

by James J. Bull, Kelly A. Christensen, Carly Scott, Benjamin R. Jack, Cameron J. Crandall and Stephen M. Krone

Antibiotics 2018, 7(1), 8; https://doi.org/10.3390/antibiotics7010008 - 29 Jan 2018

Cited by 35 | Viewed by 5518

Abstract

Bacteria growing on surfaces appear to be profoundly more resistant to control by lytic bacteriophages than do the same cells grown in liquid. Here, we use simulation models to investigate whether spatial structure per se can account for this increased cell density in [...] Read more.

Bacteria growing on surfaces appear to be profoundly more resistant to control by lytic bacteriophages than do the same cells grown in liquid. Here, we use simulation models to investigate whether spatial structure per se can account for this increased cell density in the presence of phages. A measure is derived for comparing cell densities between growth in spatially structured environments versus well mixed environments (known as mass action). Maintenance of sensitive cells requires some form of phage death; we invoke death mechanisms that are spatially fixed, as if produced by cells. Spatially structured phage death provides cells with a means of protection that can boost cell densities an order of magnitude above that attained under mass action, although the effect is sometimes in the opposite direction. Phage and bacteria self organize into separate refuges, and spatial structure operates so that the phage progeny from a single burst do not have independent fates (as they do with mass action). Phage incur a high loss when invading protected areas that have high cell densities, resulting in greater protection for the cells. By the same metric, mass action dynamics either show no sustained bacterial elevation or oscillate between states of low and high cell densities and an elevated average. The elevated cell densities observed in models with spatial structure do not approach the empirically observed increased density of cells in structured environments with phages (which can be many orders of magnitude), so the empirical phenomenon likely requires additional mechanisms than those analyzed here. Full article

(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)

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2 pages, 208 KiB

Open AccessEditorial

Acknowledgement to Reviewers of Antibiotics in 2017

by Antibiotics Editorial Office

Antibiotics 2018, 7(1), 7; https://doi.org/10.3390/antibiotics7010007 - 25 Jan 2018

Viewed by 2673

Abstract

Peer review is an essential part in the publication process, ensuring that Antibiotics maintains high quality standards for its published papers.[...] Full article

6 pages, 179 KiB

Open AccessArticle

Characteristics of Pediatric Antimicrobial Stewardship Programs: Current Status of the Sharing Antimicrobial Reports for Pediatric Stewardship (SHARPS) Collaborative

by Christopher McPherson, Brian R. Lee, Cindy Terrill, Adam L. Hersh, Jeffrey S. Gerber, Matthew P. Kronman and Jason G. Newland

Antibiotics 2018, 7(1), 4; https://doi.org/10.3390/antibiotics7010004 - 25 Jan 2018

Cited by 22 | Viewed by 5498

Abstract

In response to the growing epidemic of antibiotic-resistant bacterial infections, antimicrobial stewardship programs (ASP) have been rapidly implemented in the United States (US). This study examines the prevalence of the Centers for Disease Control and Prevention’s (CDC) seven core elements of a successful [...] Read more.

In response to the growing epidemic of antibiotic-resistant bacterial infections, antimicrobial stewardship programs (ASP) have been rapidly implemented in the United States (US). This study examines the prevalence of the Centers for Disease Control and Prevention’s (CDC) seven core elements of a successful ASP within a large subset of US Children’s Hospitals. In 2016, a survey was conducted of 52 pediatric hospitals assessing the presence of the seven core elements: leadership commitment, accountability, drug expertise, action, tracking, reporting, and education. Forty-nine hospitals (94%) had established ASPs and 41 hospitals (79%) included all seven core elements. Physician accountability (87%) and a dedicated ASP pharmacist or drug expert (88%) were present in the vast majority of hospitals. However, substantial variability existed in the financial support allotted to these positions. This variability did not predict program actions, tracking, reporting, and education. When compared with previous surveys, these results document a dramatic increase in the prevalence and resources of pediatric stewardship programs, although continued expansion is warranted. Further research is required to understand the feasibility of various core stewardship activities and the impact on patient outcomes in the setting of finite resources. Full article

(This article belongs to the Special Issue Surveillance of Antimicrobial Use and Resistance in Children)

15 pages, 730 KiB

Open AccessArticle

Reevaluation of the Acute Cystitis Symptom Score, a Self-Reporting Questionnaire. Part I. Development, Diagnosis and Differential Diagnosis

by Jakhongir F. Alidjanov, Kurt G. Naber, Ulugbek A. Abdufattaev, Adrian Pilatz and Florian M. E. Wagenlehner

Antibiotics 2018, 7(1), 6; https://doi.org/10.3390/antibiotics7010006 - 15 Jan 2018

Cited by 20 | Viewed by 5382

Abstract

This study aimed to reevaluate the Acute Cystitis Symptom Score (ACSS). The ACSS is a simple and standardized self-reporting questionnaire for the diagnosis of acute uncomplicated cystitis (AC) assessing typical and differential symptoms, quality of life, and possible changes after therapy in female [...] Read more.

This study aimed to reevaluate the Acute Cystitis Symptom Score (ACSS). The ACSS is a simple and standardized self-reporting questionnaire for the diagnosis of acute uncomplicated cystitis (AC) assessing typical and differential symptoms, quality of life, and possible changes after therapy in female patients with AC. This paper includes literature research, development and evaluation of the ACSS, an 18-item self-reporting questionnaire including (a) six questions about “typical” symptoms of AC, (b) four questions regarding differential diagnoses, (c) three questions on quality of life, and (d) five questions on additional conditions that may affect therapy. The ACSS was evaluated in 228 women (mean age 31.49 ± 11.71 years) in the Russian and Uzbek languages. Measurements of reliability, validity, predictive ability, and responsiveness were performed. Cronbach’s alpha for ACSS was 0.89, split-half reliability was 0.76 and 0.79 for first and second halves, and the correlation between them was 0.87. Mann-Whitney U test revealed a significant difference in scores of the “typical” symptoms between patients and controls (10.50 vs. 2.07, p < 0.001). The optimal threshold score was 6 points, with a 94% sensitivity and 90% specificity to predict AC. The “typical” symptom score decreased significantly when comparing before and after therapy (10.4 and 2.5, p < 0.001). The reevaluated Russian and Uzbek ACSS are accurate enough and can be recommended for clinical studies and practice for initial diagnosis and monitoring the process of the treatment of AC in women. Evaluation in German, UK English, and Hungarian languages was also performed and in other languages evaluation of the ACSS is in progress Full article

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14 pages, 3590 KiB

Open AccessArticle

Screening of E. coli β-clamp Inhibitors Revealed that Few Inhibit Helicobacter pylori More Effectively: Structural and Functional Characterization

by Preeti Pandey, Vijay Verma, Suman Kumar Dhar and Samudrala Gourinath

Antibiotics 2018, 7(1), 5; https://doi.org/10.3390/antibiotics7010005 - 11 Jan 2018

Cited by 3 | Viewed by 4832

Abstract

The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes β-clamp an important drug target. Helicobacter pylori (H. pylori) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out [...] Read more.

The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes β-clamp an important drug target. Helicobacter pylori (H. pylori) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out whether difference in DNA replication proteins behavior accounts for any difference in drug response when compared to E. coli, in the present study, we have tested E. coli β-clamp inhibitor molecules against H. pylori β-clamp. Various approaches were used to test the binding of inhibitors to H. pylori β-clamp including docking, surface competition assay, complex structure determination, as well as antimicrobial assay. Out of five shortlisted inhibitor molecules on the basis of docking score, three molecules, 5-chloroisatin, carprofen, and 3,4-difluorobenzamide were co-crystallized with H. pylori β-clamp and the structures show that they bind at the protein-protein interaction site as expected. In vivo studies showed only two molecules, 5-chloroisatin, and 3,4-difluorobenzamide inhibited the growth of the pylori with MIC values in micro molar range, which is better than the inhibitory effect of the same drugs on E. coli. Therefore, the evaluation of such drugs against H. pylori may explore the possibility to use to generate species-specific pharmacophore for development of new drugs against H. pylori. Full article

(This article belongs to the Special Issue Bacterial DNA Replication and Replication Inhibitors)

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Figure 1

Figure 1
SPR sensorgram. SPR sensorgram showing surface competition assay between HpDNA ligase and the small molecules. A qualitative analysis of the in vitro competition between DNA ligase and small molecules (present in solution) for binding to Hpβ-clamp (immobilized on the chip surface) was carried out. For the small molecules (A) 5-chloroisatin (C1); (B) 6-nitroindazole (C2) and (C) (S)-carprofen (C3), a mass of ~6 ng of Hpβ-clamp gets immobilized on the chip surface while for (D) 5-nitroindole (C4) and (E) 3,4-difluorobenzamide (C5), a mass of ~4 ng of Hpβ-clamp gets immobilized on the chip surface. The concentration of ligase was kept the same throughout each experiment with a small molecule. As the concentration of the small molecule was increased, the SPR response decreased. Full article ">Figure 2
5-chloroisatin interaction with Hpβ-clamp. (A) 2Fo-Fc map, contoured at 1σ, of 5-chloroisatin bound to Hpβ-clamp (PDB ID: 5G4Q). The alignment of the structure of the complex (green) with that of the native (orange) did not yield significant differences in the orientation of interacting residues except for I248; (B) Ligplot of the Hpβ-clamp structure near the bound 5-chloroisatin, showing the predominantly hydrophobic interactions between the protein and the inhibitor. T173 of Hpβ-clamp did form an H-bond with the inhibitor molecule; (C) Structural alignment of Hpβ-clamp (green) and Ecβ-clamp (cyan) complex with ligand 5-chloroisatin. T172 of Ecβ-clamp makes H-bond with ligand while T175 of Hpβ-clamp makes H-bond with the ligand (PDB ID: 4N95); (D) Ligplot of Ecβ-clamp complex with 5-chloroisatin showing the types of interactions between them. Full article ">Figure 3
(S)-carprofen interaction with β-clamp. (A) 2Fo-Fc map, contoured at 1σ, of (S)-carprofen bound to Hpβ-clamp (PDB ID: 5FXT). The alignment of the structure of the co-crystal of Hpβ-clamp (green) and the inhibitor (olive) with that of the native Hpβ-clamp (orange) showed almost same orientation of interacting molecules in both structures; (B) Ligplot of the Hpβ-clamp structure near the bound (S)-carprofen, showing the hydrophobic interactions between the protein and the inhibitor; (C) Structural alignment of H. pylori (green) and E. coli β-clamp (cyan) complex with ligand (S)-carprofen. T154 of Ecβ-clamp makes H-bond with the ligand apart from other hydrophobic interactions while Hpβ-clamp and ligand interactions are dominated by hydrophobic interactions; and, (D) Ligplot of Ecβ-clamp with bound ligand showing its various interactions with ligand (PDB ID: 4MJR). Full article ">Figure 4
3, 4-difluorobenzamide interaction with β-clamp. (A) 2Fo-Fc map, contoured at 1σ, of 3, 4-difluorobenzamide in complex with Hpβ-clamp. The alignment of the structure of the co-crystal of Hpβ-clamp (green) and the inhibitor (olive) with that of the native Hpβ-clamp (orange) showed differences in the orientations of residues T175, M370, K176 and I248 between the co-crystal and native structures; (B) Ligplot of the Hpβ-clamp structure near the bound 3, 4-difluorobenzamide, showing the hydrophobic interactions between the protein and the inhibitor; (C) Structural superimposition of Hpβ-clamp (green) and Ecβ-clamp (cyan) complex with ligand 3, 4-difluorobenzamide. In both the cases, the contacts are dominated by hydrophobic interactions however the orientation of ligand molecule is differentiated by a rotation of 180 degree; and, (D) Ligplot of Ecβ-clamp bound to ligand showing the hydrophobic interactions nearby (PDB ID: 4N94). Full article ">Figure 5
Structure-based sequence alignment. β-clamps of H. pylori and E. coli were compared using structure based sequence alignment. The ligand-interacting residues are highlighted (in green box). In each block, the first line shows conservation indices for positions with a conservation index above 5. The secondary structure prediction is shown in color red (alpha-helix) and blue (beta-strand). The last two lines show consensus amino acid sequence (consensus_aa) and consensus predicted secondary structures (consensus_ss). Consensus predicted secondary structure symbols: alpha-helix:h; beta-strand:e. Consensus amino acid symbols are: conserved amino acid are in uppercase and bold letter; aliphatic (I,V, L): l; aromatic (YHWF); hydrophobic (W,F,Y,M,L,I,V,A,C,T,H): h; alcohol (S,T):o; polar residues (D,E,H,K,N,Q,R,S,T): p; tiny (A,G,C,S):t; small (A,G,C,S,V,N,D,T,P):s; bulky residues (E,F,I,K,L,M,Q,R,W,Y):b; positively charged (K,R,H):+; negatively charged (D,E): -; charged (D,E,K,R,H):c. Full article ">Figure 6
Antimicrobial activities of different drugs against H. pylori. (A) Anti-H. pylori activities of different drugs (A) 5-chloroisatin (C1) and (B) 3,4-difluorobenzamide (C5) determined by applying the disk diffusion method. Petri dish with drugs containing discs showed inhibition zones for bacterial growth; (C) Anti-H. pylori activities of drugs determined as minimum inhibitory concentrations (MICs) were obtained via the dilution method. The MIC of drug C1 and C5 are 18 µM and 824 µM, respectively. The experiments were performed in triplicates. Error bars show standard deviation of the mean (Mean ± SD). Full article ">

3523 KiB

Open AccessFeature PaperArticle

Establishing a System for Testing Replication Inhibition of the Vibrio cholerae Secondary Chromosome in Escherichia coli

by Nadine Schallopp, Sarah Milbredt, Theodor Sperlea, Franziska S. Kemter, Matthias Bruhn, Daniel Schindler and Torsten Waldminghaus

Antibiotics 2018, 7(1), 3; https://doi.org/10.3390/antibiotics7010003 - 23 Dec 2017

Cited by 9 | Viewed by 5835

Abstract

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a [...] Read more.

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a single chromosome, but about 10% of species carry a split genome spanning multiple chromosomes. The best studied bacterium in this context is the human pathogen Vibrio cholerae, with a primary chromosome (Chr1) of 3 M bps, and a secondary one (Chr2) of about 1 M bps. Replication of Chr2 is under control of a unique mechanism, presenting a potential target in the development of V. cholerae-specific antibiotics. A common challenge in such endeavors is whether the effects of candidate chemicals can be focused on specific mechanisms, such as DNA replication. To test the specificity of antimicrobial substances independent of other features of the V. cholerae cell for the replication mechanism of the V. cholerae secondary chromosome, we establish the replication machinery in the heterologous E. coli system. We characterize an E. coli strain in which chromosomal replication is driven by the replication origin of V. cholerae Chr2. Surprisingly, the E. coli ori2 strain was not inhibited by vibrepin, previously found to inhibit ori2-based replication. Full article

(This article belongs to the Special Issue Bacterial DNA Replication and Replication Inhibitors)

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Figure 1

Figure 1
DNA replication in E. coli strains with oriC or ori2 driven replication. (A) Flow cytometry analyses of DNA content in rifampicin/cephalexin treated E. coli cells with DNA replication starting at oriC (strain #1; top panel) or V. cholerae ori2 (strain #16; bottom panel) [<a href="#B25-antibiotics-07-00003" class="html-bibr">25</a>]. (B) Fluorescence microscopy of E. coli cells harboring a plasmid encoding a TetR-mVenus fusion (pMA289), a FROS array insertion and either oriC (top panel; strain SM112) or ori2 (bottom panel; strain SM113). The scale bar is 2 µm. (C) Quantification of fluorescence foci per cell for microscopy shown in B (n = 700). (D) Profile of genome-wide copy numbers based on comparative genomic hybridization (CGH). Grey dots represent values of single probes for the ori2-based strain (#16) with a Loess regression (red line, F = 0.2). For comparison, the Loess regression of the oriC-based strain #1 is shown based on published data [<a href="#B29-antibiotics-07-00003" class="html-bibr">29</a>] (blue line, F = 0.2). Positions of oriC and ori2 are indicated and the genomic position as distance from oriC. Full article ">Figure 2
Transduction efficiency of gene knockouts into oriC and ori2 E. coli strains. Colonies growing on plates supplemented with kanamycin were counted after P1 transduction of KanR-cassettes inserted in dam, hapA or seqA as indicated into E. coli strains, with chromosome replication based on oriC (MG1655 (#1)) or ori2 (NZ90). Mean values of three replicates are shown in (A) with the actual numbers and standard deviation given in the table (B). Full article ">Figure 3
Copy numbers of secondary replicons in E. coli dependent on crtS. (A) Copy number of an ori2-based minichromosome in an E. coli strain without crtS (Strain NZ72) or with crtS-site insertion on the chromosome (Strain NZ140) was measured as inverse of the lag time for cells in medium with high concentrations of ampicillin (500 µg/ mL). Strains carrying an oriF-based replicon and the crtS (Strain NZ141) or no crtS on the chromosome (Strain NZ119) were used as control. Data are the mean of three biological replicates with the indicated standard deviations. (B) Copy number of an ori2-minichromosome (pMA568) analyzed by qPCR-based marker frequency analysis relative to oriC. Mean values of three biological replicates are shown with the respective standard deviations. Full article ">Figure 4
Conjugation rate of extra replicons to oriC and ori2 E. coli strains. (A) Transfer by conjugation was tested for oriC-minichromosomes carrying a crtS site (pMA200) or no crtS (pMA308) into E. coli with chromosome replication based on oriC (MG1655) or ori2 (NZ90). (B) Conjugation of oriF-based replicons with crtS (pMA206) or without (pMA899) into E. coli with oriC (MG1655), ori2 (NZ90) or oriC at the native site and ori2 at an ectopic site (#15) [<a href="#B25-antibiotics-07-00003" class="html-bibr">25</a>,<a href="#B29-antibiotics-07-00003" class="html-bibr">29</a>]. Mean values of three biological replicates are shown in (A,B) with the actual numbers and standard deviations given in the table (C). Full article ">Figure 5
Mutation-based analysis of the DnaA box within ori2. (A–C) Ori2-based minichromosomes with different mutations at the DnaA box were tested for their ability to replicate by transformation into E. coli (XL1-Blue) or DH5αλpir. The latter was used as a control because all replicons carry an oriR6K which allows replication in strains encoding the initiator Pir. Values are ratios between respective colony numbers of three biological replicates with the indicated standard deviations. Relevant sequences are shown in (D) for DnaA boxes wt (pMA87), scrambled (pMA108), inverted (pMA109), deleted (pMA110), a weak DnaA box R3 (pMA111), a medium-strength DnaA box R2 (pMA112), a 5 bp insertion to the left of the DnaA box (pMA115) or the right (pMA114) or a 5 bp deletion to the left (pMA113) or right (pMA116) as indicated. (E) Sequences found by transformation of an assembled ori2-minichromosome with a mix of sequence combinations at positions 2–5 as indicated by “N” in the Primer sequence. DnaA-box sequences from oriC in E. coli are shown for comparison (wt, R2, R3). Six sequences found in the screen are shown with the nucleotides differing from the consensus DnaA box shaded in grey. Full article ">Figure 6
Effect of vibrepin on ori2-dependent replication. Lag time (time until OD = 0.1 was reached) is shown as the mean value of three replicates, with the respective standard deviations, in medium without vibrepin (blue) or with vibrepin (red). Vibrepin concentrations were 16 μg/mL for E. coli strains (A–B) and 1.6 μg/mL for V. cholerae strains (C). Analyzed E. coli strains replicated their chromosome based on oriC (MG1655) or ori1 (NZ90) (A) or carried an extra minireplicon with ori2 (pMA100), oriC (pMA106) or oriF (pMA129) [<a href="#B38-antibiotics-07-00003" class="html-bibr">38</a>] (B) as indicated. V. cholerae strains are the standard two-chromosome strain N16961 [<a href="#B6-antibiotics-07-00003" class="html-bibr">6</a>], an engineered derivative of N16961 with fused chromosomes (MCH1) [<a href="#B36-antibiotics-07-00003" class="html-bibr">36</a>] or a natural isolate with fused chromosomes (NSCV1) [<a href="#B37-antibiotics-07-00003" class="html-bibr">37</a>]. Full article ">Figure 7
Construction and characterization of strain NZ138. (A) Scheme of NZ138 construction. Genes are indicated by arrows, origins of replication and the truncated end of mnmG by blocks. Genes of V. cholerae are colored blue, genes of E. coli orange, resistance genes red, FRT-sites black and origins of replication yellow. Green rectangles indicate BsaI and BpiI restriction sites. Binding sites of oligonucleotides are indicated in purple, numbers in brackets are genome positions of 5’-ends of the binding sites. Genomic and linearized DNA is indicated by grey lines, plasmids by circles. (B) Growth, DNA content and protein content of NZ138 compared to MG1655 and NZ90. All strains were grown in LB medium. For growth curves, five replicates for each strain were grown in a 96-well plate at 37 °C. For determination of DNA and protein content, samples were taken in exponential phase and fixed with ethanol. The samples were split, stained with SYTOX Green (DNA) or FITC (protein) and analyzed by flow cytometry. Full article ">

2019 KiB

Open AccessArticle

Evidence for Anti-Pseudogymnoascus destructans (Pd) Activity of Propolis

by Soumya Ghosh, Robyn McArthur, Zhi Chao Guo, Rory McKerchar, Kingsley Donkor, Jianping Xu and Naowarat Cheeptham

Antibiotics 2018, 7(1), 2; https://doi.org/10.3390/antibiotics7010002 - 21 Dec 2017

Cited by 6 | Viewed by 6494

Abstract

White-nose syndrome (WNS) in bats, caused by Pseudogymnoascus destructans (Pd), is a cutaneous infection that has devastated North American bat populations since 2007. At present, there is no effective method for controlling this disease. Here, we evaluated the effect of propolis [...] Read more.

White-nose syndrome (WNS) in bats, caused by Pseudogymnoascus destructans (Pd), is a cutaneous infection that has devastated North American bat populations since 2007. At present, there is no effective method for controlling this disease. Here, we evaluated the effect of propolis against Pd in vitro. Using Sabouraud dextrose agar (SDA) medium, approximately 1.7 × 10⁷ conidia spores of the Pd strain M3906-2/mL were spread on each plate and grown to form a consistent lawn. A Kirby–Bauer disk diffusion assay was employed using different concentrations of propolis (1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%), in plates incubated at 8 °C and 15 °C. At 8 °C and 15 °C, as the concentration of propolis increased, there was an increasing zone of inhibition (ZOI), reaching the highest degree at 10% and 25% concentrations, respectively. A germule suppression assay showed a similar effect on Pd conidia germination. A MALDI-TOF-MS analysis of propolis revealed multiple constituents with a potential anti-Pd activity, including cinnamic acid, p-coumaric acid, and dihydrochalcones, which could be further tested for their individual effects. Our study suggests that propolis or its individual constituents might be suitable products against Pd. Full article

(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Anti-Pd activity of propolis. (A) Images (i–xi) and (xii–xxii) indicate the activity of propolis at 8 °C and 15 °C, respectively. The black arrowheads indicate the zone of inhibition of Pd when treated with different concentrations of propolis in comparison to water and anhydrous ethanol treatments; (B) diameter of the zones of inhibition at 8 °C and 15 °C. The error bars are standard deviations of the diameters. Full article ">Figure 2
Germule suppression assay. (A,B) represent the Pd germination assay for treatments with water, anhydrous ethanol, and various concentrations of propolis at 8 °C and 15 °C, respectively. The white arrowheads indicate the mycelial extension of the Pd spores at two different incubation temperatures. The black arrowheads indicate the inhibition of the Pd spores on exposure to propolis at different concentrations. The green arrowheads indicate the formation of white Pd lawns resulting from the treatment of spores with water or anhydrous ethanol. Full article ">Figure 3
Micrographs of Pd spores displayed at 10× and 40× magnification: (i–ii) elliptical shape of untreated Pd spores; (iii–iv) deformed Pd spores treated with 1% propolis. Full article ">Figure 4
(A) MALDI-TOF-MS of a propolis sample at a mass range of 100–400 Da. Each of the peaks on the mass spectrum represents a distinctive compound in our propolis sample. The numbers above the peaks correspond to the compounds listed in <a href="#antibiotics-07-00002-t001" class="html-table">Table 1</a>; (B) magnified version of the mass spectrum at a mass range of the 100–225 Da. Full article ">

2331 KiB

Open AccessArticle

Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation

by Ziyad Al-Kass, Joachim Spergser, Christine Aurich, Juliane Kuhl, Kathrin Schmidt, Anders Johannisson and Jane M. Morrell

Antibiotics 2018, 7(1), 1; https://doi.org/10.3390/antibiotics7010001 - 21 Dec 2017

Cited by 12 | Viewed by 7074

Abstract

Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. [...] Read more.

Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates per male). Aliquots of extended semen were used for MSLC with Equicoll, resulting in four treatment groups: control and MSLC in extender with antibiotics (CA and SA, respectively); control and MSLC in extender without antibiotics (CW and SW, respectively). Sperm motility, membrane integrity, mitochondrial membrane potential and chromatin integrity were evaluated daily by computer-assisted sperm analysis (CASA) and flow cytometry. There were no differences in sperm quality between CA and CW, or between SA and SW, although progressive motility was negatively correlated to total bacterial counts at 0 h. However, MSLC groups showed higher mean total motility (P < 0.001), progressive motility (P < 0.05), membrane integrity (P < 0.0001) and mitochondrial membrane potential (P < 0.05), as well as better chromatin integrity (P < 0.05), than controls. Sperm quality remained higher in the MSLC groups than controls throughout storage. These results indicate that sperm quality was not adversely affected by the presence of antibiotics but was improved considerably by MSLC. Full article

(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)

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