Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance.

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOBP5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.

Elucidation of the Gemcitabine Transporters of Escherichia coli K-12 and Gamma-Proteobacteria Linked to Gemcitabine-Related Chemoresistance.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a widely used anticancer drug, is considered a gold standard in treating aggressive pancreatic cancers. Gamma-proteobacteria that colonize the pancreatic tumors contribute to chemoresistance against gemcitabine by metabolizing the drug to a less active and deaminated form. The gemcitabine transporters of these bacteria are unknown to date. Furthermore, there is no complete knowledge of the gemcitabine transporters in Escherichia coli or any other related proteobacteria. In this study, we investigate the complement of gemcitabine transporters in E. coli K-12 and two common chemoresistance-related bacteria (Klebsiella pneumoniae and Citrobacter freundii). We found that E. coli K-12 has two high-affinity gemcitabine transporters with distinct specificity properties, namely, NupC and NupG, whereas the gemcitabine transporters of C. freundii and K. pneumoniae include the NupC and NupG orthologs, functionally indistinguishable from their counterparts, and, in K. pneumoniae, one additional NupC variant, designated KpNupC2. All these bacterial transporters have a higher affinity for gemcitabine than their human counterparts. The highest affinity (KM 2.5-3.0 µΜ) is exhibited by NupGs of the bacteria-specific nucleoside-H+ symporter (NHS) family followed by NupCs (KM 10-13 µΜ) of the concentrative nucleoside transporter (CNT) family, 15-100 times higher than the affinities reported for the human gemcitabine transporter hENT1/SLC29A1, which is primarily associated with gemcitabine uptake in the pancreatic adenocarcinoma cells. Our results offer a basis for further insight into the role of specific bacteria in drug availability within tumors and for understanding the structure-function differences of bacterial and human drug transporters.

Multiplex lateral flow test strip for detection of carbapenemase genes using barcoded tetrahedral DNA capture probe-based biosensing interface.

Carbapenem-resistant Enterobacterales pose significant global health challenges due to their rapid spread and ability to hydrolyse various beta-lactam antibiotics. Rapid tests for these carbapenemase genes are crucial to ensure appropriate prescription administration and infection control. In this study, we developed a rapid visual nanodiagnostic platform for multiplexed detection of carbapenemase genes using a lateral flow strip. The nanodiagnostic strip was designed with separate barcoded DNA tetrahedrons for the blaKPC and blaNDM genes. These tetrahedrons were distributed on a nitrocellulose membrane at two different test lines as capture probes. When tested against a panel of carbapenemase genes, the tetrahedral probes captured single-stranded amplicons of asymmetric PCR via strand hybridisation. The amplicons acted as bridging elements, binding the DNA-modified gold nanoparticles to the test line of the strip, resulting in clear visual readouts specific to the blaKPC and blaNDM genes. By employing barcoded tetrahedrons and asymmetric PCR in conjunction with the lateral flow strip, a single diagnostic test enabled the detection of multiple carbapenemase genes. The test yielded results as low as 0.12 fM for blaKPC and 0.05 fM for blaNDM within 75 min. Furthermore, the strip effectively identified specific carbapenemase genes in clinical isolates using real-time PCR, antibody-based lateral flow systems for carbapenemase detection, and carbapenemase phenotype experiments. Thus, the strip develop has a high potential for testing blaKPC and blaNDM genes in practice.

Colonization of White-Tailed Deer (Odocoileus virginianus) from Urban and Suburban Environments with Cephalosporinase- and Carbapenemase-Producing Enterobacterales.

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance (n = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P < 0.001), male (OR = 1.8, P = 0.001) deer from urbanized habitats (OR = 1.4, P = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring blaKPC-2 and an Escherichia coli harboring blaNDM-5, from two deer from urban habitats. The genetic landscape of the plasmid carrying blaKPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying blaNDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the blaNDM-5, has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.

Imipenem-Relebactam Susceptibility and Genotypic Characteristics of Carbapenem-Resistant Enterobacterales Identified during Population-Based Surveillance.

Laboratories submit all carbapenem-resistant Enterobacter, Escherichia coli, and Klebsiella species to the Alameda County Public Health Department (ACPHD). ACPHD evaluated 75 isolates submitted during 9 months for susceptibility to imipenem-relebactam (I-R) and, using whole-genome sequencing, identified ß-lactamase genes. Of 60 (80%) isolates susceptible to I-R, 8 (13%) had detectable carbapenemase genes, including 4 KPC, two NDM, and two OXA-48-like; we described the relationship between the presence of ß-lactamase resistance genes and susceptibility to I-R.

Evaluation of the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in positive blood culture broths.

The rapid detection of extended-spectrum ß-lactamase Enterobacterales (ESBL-E) in a positive blood culture is important in order to initiate an appropriate antibiotic therapy and thus decrease mortality. We evaluated the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in 100 positive blood culture broths to detect (in ten minutes) the presence or absence of ESBL-E. The BL-RED test appears to be an easy, rapid and reliable test to detect the presence of ESBL directly in Gram negative bacilli-positive blood culture broths, with good performances (sensibility =97.3%, specificity =90.5%, predictive positive value =85.7% and predictive negative value =98.3%). This test could be useful for therapeutic decisions and adjustments of sepsis empirical antibiotic therapy, particularly in wards where the ecology is unfavorable, such as in intensive care units.

Activity of temocillin against ESBL-, AmpC-, and/or KPC-producing Enterobacterales isolated in Poland.

We evaluated the in vitro effectiveness of temocillin and several commonly used antimicrobials against Enterobacterales bacteria in isolates from Polish patients. We tested 400 isolates: 260 extended-spectrum ß-lactamase (ESBL)- and/or ampC ß-lactamase (AmpC)-producing isolates; 40 Klebsiella pneumoniae carbapenemase (KPC)-producing isolates; and 100 ESBL-, AmpC-, and KPC-negative isolates. The minimal inhibitory concentrations (MICs) of temocillin and 16 other antimicrobials were determined by reference microdilution. We also determined the activities of fosfomycin and ceftazidime/avibactam in KPC-producing isolates. The antibiotic sensitivities were interpreted according to EUCAST, BSAC, and CLSI criteria. Overall, 91% of the isolates were susceptible to temocillin using the urinary tract infection breakpoint (≤ 32 mg/L), and 61.8% were susceptible using the systemic infection breakpoint (≤ 8 mg/L). Meropenem and imipenem were the most active drugs (MIC50 values of 0.06 and 0.5 mg/L, respectively). Colistin and ertapenem (both MIC50 = 0.12 mg/L) were less active than meropenem or imipenem, but some strains were 77% susceptible to each of them. Among the KPC-producing isolates, 42.5% had MIC values of ≤ 32 mg/L (urinary tract infection breakpoint), but 100% were resistant to temocillin (systemic infection breakpoint). Ceftazidime/avibactam was active against 100% of the KPC-producing isolates, and fosfomycin was active against 40%. The empirical susceptibility rate observed among the urinary isolates suggests that temocillin may be considered as an alternative to carbapenems in the absence of KPC-producing bacteria. With regard to isolates from other sources, temocillin might be useful as a documented therapy agent or an empirical treatment in hospitals with a low prevalence of ESBL/AmpC-producing strains.

Microbial diversity and co-occurrence patterns in deep soils contaminated by polycyclic aromatic hydrocarbons (PAHs).

Numerous studies have enriched our knowledge of the microbial community composition and metabolic versatility of contaminated soil. However, there remains a substantial gap regarding the bioassembly patterns of the indigenous microbial community distribution in contaminated deep soils. Herein, the indigenous microbial community structure diversity, function, and co-occurrence relationships in aged PAH-contaminated deep soil collected from an abandoned chemical facility were investigated using high-throughput sequencing. The results showed that the dominant phyla in all samples were responsible for PAH degradation and included Proteobacteria (20.86%-81.37%), Chloroflexi (2.03%-28.44%), Firmicutes (3.06%-31.16%), Actinobacteria (2.92%-11.91%), Acidobacteria (0.41%-12.68%), and Nitrospirae (0.81%-9.21%). Eighty biomarkers were obtained by linear discriminant analysis of effect size (LEfSe), and most of these biomarkers were PAH degraders. Functional predictions using Tax4Fun indicated that the aged contaminated soil has the potential for PAH degradation. Statistical analysis showed that in contrast with the PAH concentration, edaphic properties (nutrients and pH) were significantly correlated (r > 0.25, P < 0.01) with the bacterial community and functional composition. Co-occurrence network analysis (modularity index of 0.781) revealed non-random assembly patterns of the bacterial communities in the PAH-contaminated soils. The modules in the network were mainly involved in carbon and nitrogen cycles, organic substance degradation, and biological electron transfer processes. Microbes from the same module had strong ecological linkages. Additionally, SAR202 clade, Thermoanaerobaculum, Nitrospira, and Xanthomonadales, which were identified as keystone species, played an irreplaceable role in the network. Overall, our results suggested that environmental factors such as nutrients and pH, together with ecological function, are the main factors driving the assembly of microbial communities in aged PAH-contaminated deep soils.

Response of particle-associated bacteria to long-term heavy metal contamination in a tropical estuary.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > ß-glucosidase > aminopeptidase, while it was in the order of ß-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

ZHO-1, an intrinsic MBL from the environmental Gram-negative species Zhongshania aliphaticivorans.

OBJECTIVES: Our aim was to characterize the putative MBL of the environmental strain Zhongshania aliphaticivorans isolated from a marine environment. METHODS: The putative MBL was identified in silico using the NCBI database. The ß-lactamase gene was cloned into different Escherichia coli backgrounds. Kinetic parameters were determined using the purified enzyme. RESULTS: The enzyme named ZHO-1 shared 51% amino acid identity with the acquired class B carbapenemases IMP-1, KHM-1 and DIM-1. Expression of the blaZHO-1 gene in a susceptible E. coli resulted in a carbapenemase phenotype. Kinetic parameters determined from purified ZHO-1 enzyme showed that it had significant hydrolytic activity against most ß-lactams including penicillins, cephalosporins and carbapenems, with the exception of aztreonam and cefepime. CONCLUSIONS: This study adds to the knowledge regarding environmental species as a reservoir of possible clinically relevant MBLs.

Direct antimicrobial susceptibility testing from the blood culture pellet obtained for MALDI-TOF identification of Enterobacterales and Pseudomonas aeruginosa.

To standardize the methodology for conducting direct antimicrobial susceptibility testing (AST) of Enterobacterales and Pseudomonas aeruginosa causing bacteremia from positive blood culture pellets. Two methods for processing positive blood cultures with Enterobacterales and P. aeruginosa were compared: a conventional method for identification and AST versus a direct method obtaining a pellet for both matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) identification and direct AST. A total of 157 (145 Enterobacterales, 12 P. aeruginosa) positive blood cultures were included. Microorganism identification showed 100% concordance between both methods at species and genus level. Definitive AST results were obtained 24 h earlier with the rapid method than the conventional one (p < 0.001). Of the 2814 MICs generated, there were discrepancies with respect to the conventional method in 47 (1.7%), 0.3% being very major (VME) and 1.3% major (ME) errors. Better results for AST were obtained when colony counts with the pellet were ≥ 105 cfu/ml. The essential agreement (EA) for antibiotics tested in Enterobacterales was at least 97%, except for ampicillin (95%). Regardless of colony count, the greatest discrepancies were observed for first/s-generation cephalosporins and aminoglycosides. In P. aeruginosa, EA was at least 92%, except for piperacillin-tazobactam (84%) and cefepime (76%). No VME occurred except for ceftazidime (8%). ME occurred in piperacillin/tazobactam (16%), ticarcillin, ceftazidime, tobramycin, amikacin, and colistin (8% each). Direct use of the blood culture pellet permits fast AST in bacteremia of Enterobacterales, enabling the clinicians to perform an early treatment adjustment. However, for Pseudomonas aeruginosa, the data needs expanding to improve the reliability of this technique.

Broad-spectrum antimicrobial activity by Burkholderia cenocepacia TAtl-371, a strain isolated from the tomato rhizosphere.

The Burkholderia cepacia complex (Bcc) comprises a group of 24 species, many of which are opportunistic pathogens of immunocompromised patients and also are widely distributed in agricultural soils. Several Bcc strains synthesize strain-specific antagonistic compounds. In this study, the broad killing activity of B. cenocepacia TAtl-371, a Bcc strain isolated from the tomato rhizosphere, was characterized. This strain exhibits a remarkable antagonism against bacteria, yeast and fungi including other Bcc strains, multidrug-resistant human pathogens and plant pathogens. Genome analysis of strain TAtl-371 revealed several genes involved in the production of antagonistic compounds: siderophores, bacteriocins and hydrolytic enzymes. In pursuit of these activities, we observed growth inhibition of Candida glabrata and Paraburkholderia phenazinium that was dependent on the iron concentration in the medium, suggesting the involvement of siderophores. This strain also produces a previously described lectin-like bacteriocin (LlpA88) and here this was shown to inhibit only Bcc strains but no other bacteria. Moreover, a compound with an m/z 391.2845 with antagonistic activity against Tatumella terrea SHS 2008T was isolated from the TAtl-371 culture supernatant. This strain also contains a phage-tail-like bacteriocin (tailocin) and two chitinases, but the activity of these compounds was not detected. Nevertheless, the previous activities are not responsible for the whole antimicrobial spectrum of TAtl-371 seen on agar plates, suggesting the presence of other compounds yet to be found. In summary, we observed a diversified antimicrobial activity for strain TAtl-371 and believe it supports the biotechnological potential of this Bcc strain as a source of new antimicrobials.

Aliphatic Hydrocarbon Enhances Phenanthrene Degradation by Autochthonous Prokaryotic Communities from a Pristine Seawater.

The microbial diversity and functioning around oceanic islands is poorly described, despite its importance for ecosystem homeostasis. Here, we aimed to verify the occurrence of microbe-driven phenanthrene co-oxidation in the seawater surrounding the Trindade Island (Brazil). We also used Next-Generation Sequencing to evaluate the effects of aliphatic and polycyclic aromatic hydrocarbons (PAHs) on these microbial community assemblies. Microcosms containing seawater from the island enriched with either labelled (9-14C) or non-labelled phenanthrene together with hexadecane, weathered oil, fluoranthene or pyrene, and combinations of these compounds were incubated. Biodegradation of phenanthrene-9-14C was negatively affected in the presence of weathered oil and PAHs but increased in the presence of hexadecane. PAH contamination caused shifts in the seawater microbial community-from a highly diverse one dominated by Alphaproteobacteria to less diverse communities dominated by Gammaproteobacteria. Furthermore, the combination of PAHs exerted a compounded negative influence on the microbial community, reducing its diversity and thus functional capacity of the ecosystem. These results advance our understanding of bacterial community dynamics in response to contrasting qualities of hydrocarbon contamination. This understanding is fundamental in the application and monitoring of bioremediation strategies if accidents involving oil spillages occur near Trindade Island and similar ecosystems.

Comparative study of antiestrogenic activity of two dyes after Fenton oxidation and biological degradation.

In present study, two methods (Fenton oxidation and biological degradation) were used to degrade azo dye (Reactive Black 5, RB5) and anthraquinone dye (Remazol Brilliant Blue R, RBBR). The changes of antiestrogenic activities of these two dyes through two degradation methods were detected using the yeast two-hybrid assay method. Fluorescence spectroscopy together with gas chromatography-mass spectrometry (GC-MS) method was performed to analyze the metabolites of RB5 and RBBR after Fenton oxidation and biological degradation. Results indicated that by Fenton oxidation, the decolorization of RB5 and RBBR were 99.31% and 96.62%, respectively, which were much higher than that by biological degradation. Dissolved organic carbon (DOC) reduction rates of RB5 and RBBR after Fenton oxidation were also much higher than that after biological degradation. By Fenton oxidation, the antiestrogenic activities of RB5 and RBBR all decreased below detection limit after degradation, while by biological degradation all of them increased significantly after degradation. Fluorescence spectroscopy analysis and GC-MS analysis confirmed the degradation effects of RB5 and RBBR by these two degradation methods. In addition, fluorescence spectroscopy analysis revealed that the metabolites humic acid-like substances might contribute to the increasing of antiestrogenic activity of RB5 and RBBR after biological degradation.

Wohlfahrtiimonas chitiniclastica: current insights into an emerging human pathogen.

Since the first description of Wohlfahrtiimonas chitiniclastica in 2008, a number of well described case reports demonstrating its pathogenic role in humans have been published. Infections may be closely linked to flies, such as Wohlfahrtia magnifica, Lucilia sericata, Chrysomya megacephala or Musca domestica. These insects are potent vectors for the distribution of W. chitiniclastica causing local or systemic infections originating from wounds infested with fly larvae. However, other potential sources of transmission of W. chitiniclastica have been described such as soil or chicken meat. Infections in humans reported to date comprise wound infections, cellulitis, osteomyelitis and sepsis. This review summarizes all the literature available up to now and gives the current knowledge about this emerging human pathogen. Additionally, four patients with proven W. chitiniclastica infections treated at Dresden University Hospital between 2013 and 2015, are included. Special focus was placed on microbiological identification and antibiotic susceptibility testing of the pathogen.

Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2(-)) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (ßAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of ßAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of ßAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of ßAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

Sulfonamide inhibition studies of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2, TcruCA.

We report a sulfonamide/sulfamate inhibition study of the α-carbonic anhydrase (CA, EC 4.2.1.1) present in the gammaproteobacterium Thiomicrospira crunogena XCL-2, a mesophilic hydrothermal vent-isolate organism, TcruCA. As Thiomicrospira crunogena is one of thousands of marine organisms that uses CA for metabolic regulation, the effect of sulfonamide inhibition has been considered. Sulfonamide-based drugs have been widely used in a variety of antibiotics, and bioelimination of these compounds results in exposure of these compounds to marine life. The enzyme was highly inhibited, with Ki values ranging from 2.5 to 40.7nM by a variety of sulfonamides including acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide and benzenesulfonamides incorporating 4-hydroxyalkyl moieties. Less effective inhibitors were topiramate, zonisamide, celecoxib, saccharin and hydrochlorothiazide as well as simple benzenesulfonamides incorporating amino, halogeno, alkyl, aminoalkyl and other moieties in the ortho- or para-positions of the aromatic ring (Kis of 202-933nM). The active site interactions between TcruCA and three clinically-used CA inhibitors, acetazolamide (Diamox®), dorzolamide (Trusopt®), and brinzolamide (Azopt®) are studied using molecular docking to provide insight into the reported Ki values. Comparison between various enzymes belonging to this family may also bring interesting hints in these fascinating phenomena.

Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary between regions of the human gastrointestinal tract.

PURPOSE: Vitamin D is well known for its effects on bone mineralisation but has also been attributed immunomodulatory properties. It positively influences human health, but in vivo data describing vitamin D effects on the human gut microbiome are missing. We aimed to investigate the effects of oral vitamin D3 supplementation on the human mucosa-associated and stool microbiome as well as CD8(+) T cells in healthy volunteers. METHODS: This was an interventional, open-label, pilot study. Sixteen healthy volunteers (7 females, 9 males) were endoscopically examined to access a total of 7 sites. We sampled stomach, small bowel, colon, and stools before and after 8 weeks of vitamin D3 supplementation. Bacterial composition was assessed by pyrosequencing the 16S rRNA gene (V1-2), and CD8(+) T cell counts were determined by flow cytometry. RESULTS: Vitamin D3 supplementation changed the gut microbiome in the upper GI tract (gastric corpus, antrum, and duodenum). We found a decreased relative abundance of Gammaproteobacteria including Pseudomonas spp. and Escherichia/Shigella spp. and increased bacterial richness. No major changes occurred in the terminal ileum, appendiceal orifice, ascending colon, and sigmoid colon or in stools, but the CD8(+) T cell fraction was significantly increased in the terminal ileum. CONCLUSION: Vitamin D3 modulates the gut microbiome of the upper GI tract which might explain its positive influence on gastrointestinal diseases, such as inflammatory bowel disease or bacterial infections. The local effects of vitamin D demonstrate pronounced regional differences in the response of the GI microbiome to external factors, which should be considered in future studies investigating the human microbiome.

Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.).