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Asset Tag: Bacterial

April 21, 2020

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon.

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non-encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64-256 µg ml-1 ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 µg ml-1 ) and colistin (MIC 256 µg ml-1 ) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high-level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC. © 2018 John Wiley & Sons Ltd.

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April 21, 2020

Engineering and modification of microbial chassis for systems and synthetic biology.

Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The ‘Minimum genome factory’ (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The ‘cell factory’ project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the ‘secretome’ [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter’s group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.

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April 21, 2020

BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage ? infection, induction of ? prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

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April 21, 2020

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation.

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp. Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10-2 and 3.7 ×10-2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria.

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April 21, 2020

Cytotoxic and Antibacterial Cervinomycins B1-4 from a Streptomyces Species.

AntiSMASH analysis of genome DNA of Streptomyces CPCC 204980, a soil isolate with potent antibacterial activity, revealed a gene cluster for polycyclic xanthones. A subsequent chemical study confirmed that the microorganism produced polycyclic xanthone cervinomycin A2 (1) and the new congeners cervinomycins B1-4 (2-5). The structures of 1-5 were determined by comprehensive analyses of MS and NMR data, which indicated that 2-5 featured a common dihydro-D ring in the polycyclic xanthone core moiety of their molecules. 2-5 are toxic to human cancer cells and active against Gram-positive bacteria.

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April 21, 2020

Gramella fulva sp. nov., isolated from a dry surface of tidal flat.

A novel Gram-stain-negative, aerobic, motile by means of gliding, and short rod-shaped bacterium, designated strain SH35T, was isolated from the dry surface of a tidal flat in Hwasung-si, South Korea. Growth occurred at 10-40°C (optimum 30°C), at pH 6.0-8.0 (optimum pH 7.0), in 1-12% NaCl (optimum 2%), and was inhibited in the absence of NaCl and Ca2+ ions. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SH35T belonged to the genus Gramella and was a member of the family Flavobacteriaceae with highest sequence similarity to Gramella flava JLT2011T (96.1%), followed by Gramella oceani CCAMSZ-TT (95.6%), and 93.0-94.9% to other recognized Gramella species. The major cellular fatty acids (> 5% of the total) of strain SH35T were iso-C15:0, Iso-C16:0, anteiso-C15:0, iso-C17:0 3-OH and summed feature 9 (C16:0 10-methyl and/or C17:1 iso ?9?). The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and nine unidentified polar lipids. The major respiratory quinone and the predominant polyamine were menaquinone-6 (MK-6) and symhomospermidine, respectively. The DNA G + C content was 40.5 mol% (39.7% based on total genome calculations). Based on phylogenetic analysis and physiological and biochemical characterization, strain SH35T represents a novel species of the genus Gramella, for which the name Gramella fulva sp. nov. is proposed. The type strain is SH35T (= KACC 19447T = JCM 32369T).

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April 21, 2020

Complete Genome Sequence of Saccharospirillum mangrovi HK-33T Sheds Light on the Ecological Role of a Bacterium in Mangrove Sediment Environment.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911 bp as well as an average G?+?C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.

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April 21, 2020

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the “slippery liquid-infused porous surfaces” (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

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April 21, 2020

Antarctic heterotrophic bacterium Hymenobacter nivis P3T displays light-enhanced growth and expresses putative photoactive proteins.

Hymenobacter nivis P3T is a heterotrophic bacterium isolated from Antarctic red snow generated by algal blooms. Despite being non-photosynthetic, H. nivis was dominantly found in the red snow environment that is exposed to high light and UV irradiation, suggesting that this species can flourish under such harsh conditions. In order to further understand the adaptive strategies on the snow surface environment of Antarctica, the genome of H. nivis P3T was sequenced and analyzed, which identified genes putatively encoding for light-reactive proteins such as proteorhodopsin, phytochrome, photolyase and several copies of cryptochromes. Culture-based experiments revealed that H. nivis P3T growth was significantly enhanced under light conditions, while dark conditions had increased extracellular polymeric substances. Furthermore, the expression of several putative light-reactive proteins was determined by proteomic analysis. These results indicate that H. nivis P3T is able to potentially utilize light, which may explain its dominance on the red snow surface environment of Antarctica. ORIGINALITY-SIGNIFICANCE STATEMENT: The role of proteorhodopsin in heterotrophic bacteria is not well-characterized, as only a handful of proteorhodopsin-harbouring isolates were shown to have a light-enhanced phenotype through culture-based experiments to date. This is the first study that demonstrates light-stimulated growth and protein expression evidence of photoactive proteins for a non-marine psychrophile and for a member of the genus Hymenobacter. It is also the first study that provides comprehensive proteome information for this genus. This study presents significant results in understanding the adaptive mechanism of a heterotrophic non-photosynthetic bacterium thriving on the snow surface environment of Antarctica as well as demonstrating the role of light-utilization in promoting growth, possibly through proteorhodopsin. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

New Delhi metallo-ß-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.Testing included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.The complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for ß-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of  E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.This is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual ß-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.

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April 21, 2020

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Geography Shapes the Population Genomics of Salmonella enterica Dublin.

Salmonella enterica serotype Dublin (S. Dublin) is a bovine-adapted serotype that can cause serious systemic infections in humans. Despite the increasing prevalence of human infections and the negative impact on agricultural processes, little is known about the population structure of the serotype. To this end, we compiled a manually curated data set comprising of 880 S. Dublin genomes. Core genome phylogeny and ancestral state reconstruction revealed that region-specific clades dominate the global population structure of S. Dublin. Strains of S. Dublin in the UK are genomically distinct from US, Brazilian, and African strains. The geographical partitioning impacts the composition of the core genome as well as the ancillary genome. Antibiotic resistance genes are almost exclusively found in US genomes and are mediated by an IncA/C2 plasmid. Phage content and the S. Dublin virulence plasmid were strongly conserved in the serotype. Comparison of S. Dublin to a closely related serotype, S. enterica serotype Enteritidis, revealed that S. Dublin contains 82 serotype specific genes that are not found in S. Enteritidis. Said genes encode metabolic functions involved in the uptake and catabolism of carbohydrates and virulence genes associated with type VI secretion systems and fimbria assembly respectively. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

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