Long-term evolution of antibiotic persistence in P. aeruginosa lung infections

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as persistence. While a variety of resistance mechanisms and underlying genetics are well characterised in vitro and in vivo, the evolution of persistence, and how it interacts with resistance in situ is less well understood. We assayed for persistence and resistance with three clinically relevant antibiotics: meropenem, ciprofloxacin and tobramycin, in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to forty years of evolution. We find evidence that persistence is under positive selection in the lung and that it can particularly act as an evolutionary stepping stone to resistance. However, this pattern is not universal and depends on the bacterial clone type and antibiotic used, indicating an important role for antibiotic mode of action.

Interplay between bacterial clone and plasmid in the spread of antibiotic resistance genes in the gut: lessons from a temporal study in veal calves

Intestinal carriage of extended spectrum β-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over six months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates (blaCTX-M-1 (64.7%), blaCTX-M -14 (33.5%) or blaCTX-M-15 (1.8%)) were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone vs. plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids and bacterial clones contribute to optimize fitness in specific environments.

Advancing Genetic Tools in Streptococcus pneumoniae

Streptococcus pneumoniae is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in S. pneumoniae that expand experimental applications.

Aquatic Thermal Reservoirs of Microbial Life in a Remote and Extreme High Andean Hydrothermal System

Hydrothermal systems are ideal to understand how microbial communities cope with challenging conditions. Lirima, our study site, is a polyextreme, high-altitude, hydrothermal ecosystem located in the Chilean Andean highlands. Herein, we analyze the benthic communities of three nearby springs in a gradient of temperature (42–72 °C represented by stations P42, P53, and P72) and pH, and we characterize their microbial diversity by using bacteria 16S rRNA (V4) gene metabarcoding and 16S rRNA gene clone libraries (bacteria and archaea). Bacterial clone libraries of P42 and P53 springs showed that the community composition was mainly represented by phototrophic bacteria (Chlorobia, 3%, Cyanobacteria 3%, at P42; Chlorobia 5%, and Chloroflexi 5% at P53), Firmicutes (32% at P42 and 43% at P53) and Gammaproteobacteria (13% at P42 and 29% at P53). Furthermore, bacterial communities that were analyzed by 16S rRNA gene metabarcoding were characterized by an overall predominance of Chloroflexi in springs with lower temperatures (33% at P42), followed by Firmicutes in hotter springs (50% at P72). The archaeal diversity of P42 and P53 were represented by taxa belonging to Crenarchaeota, Diapherotrites, Nanoarchaeota, Hadesarchaeota, Thaumarchaeota, and Euryarchaeota. The microbial diversity of the Lirima hydrothermal system is represented by groups from deep branches of the tree of life, suggesting this ecosystem as a reservoir of primitive life and a key system to study the processes that shaped the evolution of the biosphere.

Analytical and preparative biosynthesis of S100B recombinant protein using the pBT7-N-His vector system and preliminary studies of structure and function of this protein

С целью исследований структуры и функций белка S100B в клетке и в тканях был проведен цикл работ по оптимизации экспрессии рекомбинантного белка (рекS100B) в E. coli. Проведены процедуры аналитической экспрессии рекS100B в составе рекомбинантной плазмиды pBT7-N-His-S100B03. При SDS-ПААГЭ лизатов клонов бактерий выявлена четко экспрессирующаяся полоса в 10 кДа, которая была идентифицирована как мономерная форма белка. Перспективы исследований рекS100B связаны с потенциальным его использованием для изучения тонких молекулярных механизмов PPI взаимодействий в системе S100B/RAGE рецептор как ключевого звена передачи сигналов в клетке и организме и в качестве перспективного объекта создания диагностических систем мониторинга состояний организма в норме и при патологии связанной с нарушениями регуляции гена и/или функций S100B белка. To study structure and functions of the S100B protein in cells and tissues, a series of studies was conducted to optimize the recombinant protein (recS100B) expression in E. coli. Procedures for analytical expression of recS100B in the pBT7-N-His-S100B03 recombinant plasmid were performed. In SDS-PAGE of bacterial clone lysate, a clear 10 kDa band expression was detected, which was identified as a monomeric form of the protein. Prospects for the S100B study are related with its potential use for investigating molecular mechanisms of PPI interactions in the S100B/RAGE system as a key signal transducer in the cell and body and as a promising object for developing diagnostic systems for monitoring the body state in normal and pathological conditions associated with impaired regulation of the gene and/or functions of the S100B protein.

Range Expansion and the Origin of USA300 North American Epidemic Methicillin-Resistant Staphylococcus aureus

ABSTRACT The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation. IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone’s geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone’s spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone’s geographic spread.

Biological Production of an Integrinαvβ3 Targeting Imaging Probe and Functional Verification

The aim of the present study is to establish a bacterial clone capable of secreting an integrinαvβ3 targeting probe with bioluminescent and fluorescent activities, and to verify its specific targeting and optical activities using molecular imaging. A bacterial vector expressing a fusion of secretory Gaussia luciferase (sGluc), mCherry, and RGD (sGluc-mCherry-RGDX3; GCR), and a control vector expressing a fusion of secretoryGaussia luciferaseand mCherry (sGluc-mCherry; GC) were constructed. The GCR and GC proteins were expressed inE. coliand secreted into the growth medium, which showed an approximately 10-fold higher luciferase activity than the bacterial lysate. Successful purification of GCR and GC was achieved using the 6X His-tag method. The GCR protein bound with higher affinity to U87MG cells than CHO cells in confocal microscopy and IVIS imaging, and also showed a high affinity for integrinαvβ3 expressing tumor xenografts in anin vivoanimal model. AnE. coliclone was established to secrete an integrinαvβ3 targeting imaging probe with bioluminescent and fluorescent activities. The probe was produced feasibly and at low cost, and has shown to be useful for the assessment of angiogenesisin vitroandin vivo.

Faucicola mancuniensis gen. nov., sp. nov., isolated from the human oropharynx

An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2T, was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20–37 °C and in the presence of up to 1.5 % NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022T (94.68 %) and an uncultured, unspeciated bacterial clone (strain S12-08; 99 %). The major fatty acids were C18 : 1ω9c, C18 : 0, C16 : 0 and C16 : 1ω6c/C16 : 1ω7c. The DNA G+C content of strain GVCNT2T was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2T exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from d-glucose, melibiose, l-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae , strain GVCNT2T is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2T ( = DSM 28411T = NCIMB 14946T).

Ecophysiology of Fe-Cycling Bacteria in Acidic Sediments

ABSTRACT Using a combination of cultivation-dependent and -independent methods, this study aimed to elucidate the diversity of microorganisms involved in iron cycling and to resolve their in situ functional links in sediments of an acidic lignite mine lake. Using six different media with pH values ranging from 2.5 to 4.3, 117 isolates were obtained that grouped into 38 different strains, including 27 putative new species with respect to the closest characterized strains. Among the isolated strains, 22 strains were able to oxidize Fe(II), 34 were able to reduce Fe(III) in schwertmannite, the dominant iron oxide in this lake, and 21 could do both. All isolates falling into the Gammaproteobacteria (an unknown Dyella-like genus and Acidithiobacillus-related strains) were obtained from the top acidic sediment zones (pH 2.8). Firmicutes strains (related to Bacillus and Alicyclobacillus) were only isolated from deep, moderately acidic sediment zones (pH 4 to 5). Of the Alphaproteobacteria, Acidocella-related strains were only isolated from acidic zones, whereas Acidiphilium-related strains were isolated from all sediment depths. Bacterial clone libraries generally supported and complemented these patterns. Geobacter-related clone sequences were only obtained from deep sediment zones, and Geobacter-specific quantitative PCR yielded 8 × 105 gene copy numbers. Isolates related to the Acidobacterium, Acidocella, and Alicyclobacillus genera and to the unknown Dyella-like genus showed a broad pH tolerance, ranging from 2.5 to 5.0, and preferred schwertmannite to goethite for Fe(III) reduction. This study highlighted the variety of acidophilic microorganisms that are responsible for iron cycling in acidic environments, extending the results of recent laboratory-based studies that showed this trait to be widespread among acidophiles.

Anaerostipes butyraticus sp. nov., an anaerobic, butyrate-producing bacterium from Clostridium cluster XIVa isolated from broiler chicken caecal content, and emended description of the genus Anaerostipes

Four butyrate-producing isolates were obtained from the caecal content of a 4-week-old broiler chicken. The 16S rRNA gene sequences were determined and confirmed the close relatedness of the four isolates, which suggested that they were derived from a single bacterial clone. Phylogenetic analysis based on 16S rRNA gene sequences showed that its closest relatives were members of cluster XIVa of the Clostridium subphylum of Gram-positive bacteria and that the closest related type strain was Anaerostipes caccae L1-92T (94.5 % similarity). Similarity levels of 96–98 % with sequences from uncultured bacteria from human stool samples were observed. On the basis of morphological, biochemical and phylogenetic characteristics, this strain is assigned to a novel species in the genus Anaerostipes, for which the name Anaerostipes butyraticus sp. nov. is proposed. The type strain is 35-7T (=LMG 24724T =DSM 22094T). An emended description of the genus Anaerostipes is also provided.