Small Colonies Research Articles

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

Community-forming traits play role in effective colonization of plant-growth-promoting bacteria and improved plant growth.

Community-forming traits (CFts) play an important role in the effective colonization of plant-growth-promoting bacterial communities that influence host plants positively by modulating their adaptive functions. In this study, by considering plant-growth-promoting traits (PGPts) and community-forming traits (CFts), three communities were constructed, viz., SM1 (PGPts), SM2 (CFts), and SM3 (PGPts+CFts). Each category isolates were picked up on the basis of their catabolic diversity of different carbon sources. Results revealed a distinctive pattern in the colonization of the communities possessed with CF traits. It was observed that the community with CFts colonized inside the plant in groups or in large aggregations, whereas the community with only PGPts colonized as separate individual and small colonies inside the plant root and leaf. The effect of SM3 in the microcosm experiment was more significant than the uninoculated control by 22.12%, 27.19%, and 9.11% improvement in germination percentage, chlorophyll content, and plant biomass, respectively. The significant difference shown by the microbial community SM3 clearly demonstrates the integrated effect of CFts and PGPts on effective colonization vis-à-vis positive influence on the host plant. Further detailed characterization of the interaction will take this technology ahead in sustainable agriculture.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

Dynamics of Microcystis surface scum formation under different wind conditions: the role of hydrodynamic processes at the air-water interface.

Due to climate change, Microcystis blooms occur at increasing frequencies in aquatic ecosystems worldwide. Wind-generated turbulence is a crucial environmental stressor that can vertically disperse the Microcystis surface scum, reducing its light availability. Yet, the interactions of Microcystis scum with the wind-generated hydrodynamic processes, particularly those at the air-water interface, remain poorly understood. Here, we explore the response of Microcystis (including colony size and migration dynamics) to varying magnitudes and durations of intermittent wind disturbances in a mesocosm system. The flow velocities, size of Microcystis colonies, and the areal coverage of the water surface by scum were measured through video observations. Our results demonstrate that low wind speeds increase colony size by providing a stable condition where Microcystis forms a scum layer and aggregates into large colonies at the air-water interface. In contrast, wind disturbances disperse scum and generate turbulence, resulting in smaller colonies with higher magnitudes of wind disturbance. We observed that surface scum can form rapidly following a long period (6h) of high-magnitude (4.5m s-1) wind disturbance. Furthermore, our results indicate reduced water surface tension caused by the presence of Microcystis, which can decrease surface flow velocity and counteract wind-driven mixing. The reduced surface tension may also drive lateral convection at the water surface. These findings suggest that Microcystis reduces surface tension, likely by releasing surface-active materials, as an adaptive response to various wind conditions. This could result in an increased rate of surface scum re-formation under wind conditions and potentially facilitate the lateral expansion of scum patches during weak wind periods. This study reveals new insights into how Microcystis copes with different wind conditions and highlights the importance of the air-water interface for Microcystis scum dynamics.

Dual-Responsive Nanogels with Cascaded Gentamicin Release and Lysosomal Escape to Combat Intracellular Small Colony Variants for Peritonitis and Sepsis Therapies.

Intracellular bacteria are the major cause of serious infections including sepsis and peritonitis, but face great challenges in fighting against the stubborn intracellular small colony variants (SCVs). Herein, the authors have developed nanogels (NGs) to destroy both planktonic bacteria and SCVs and eliminate excessive inflammations for peritonitis and sepsis therapies. Free gentamicin (GEN) and hydroxyapatite nanoparticles (NPs) with GEN loading and mannose grafts (mHAG) are inoculated into ε-polylysine NGs to obtain NG@G1-mHAG2 through crosslinking with phenylboronic acid and tannic acid. The H2O2 consumption after reaction with phenylboronic esters and the elimination of free radicals by tannic acid alleviates the escalated inflammatory status to promote sepsis therapy. After mannose-mediated uptake into macrophages, the acid-triggered degradation of mHAG NPs generates Ca2+ to destabilize lysosomes and the efficient lysosomal escape leads to reversion of hypometabolic SCVs into normal phenotype and their sensitivity to GEN. In a peritonitis mouse model, NG@G1-mHAG2 treatment provides strong and persistent bactericidal effects against both extracellular bacteria and intracellular SCVs and extends survival of peritonitis mice without apparent hepatomegaly, splenomegaly, pulmonary edema, and inflammatory cell infiltration. Thus, this study demonstrates a concise and versatile strategy to eliminate SCVs and relieve inflammatory storms for peritonitis and sepsis therapies without infection recurrence.

Staphylococcus aureus small colony variants: A potentially underestimated microbiological challenge in peritoneal dialysis

IntroductionPeritonitis remains the major infectious complication in the setting of peritoneal dialysis (PD). Despite known only moderate pathogenicity, the most frequently detected pathogens in PD-related peritonitis are surprisingly coagulase-negative staphylococci. However, this could be explained, at least in part, by Staphylococcus aureus small colony variants (SCVs) induced by PD fluids (PDFs) and misidentified by routinely used microbiological methods. Material and methodsBacteria were exposed to commonly used PDFs in various regimens designed to simulate daily use as closely as possible. Wild-type isolates and SCVs were subsequently used to determine minimum inhibitory concentrations (MICs), in vitro biofilm formation capacities, and auxotrophies. Underlying genetic alterations were investigated using whole-genome sequencing, and various microbial identification methods were tested to determine their performance for wild-types and SCVs. ResultsStable SCVs could be isolated most successfully after exposure to glucose-containing PDFs alone. The reading of MICs was significantly affected by the reduced growth of SCVs, resulting in lower MIC values in 44% of all tests. Nonsynonymous mutations were found in all but one SCV, while only two isolates showed typical auxotrophic responses. While MALDI-TOF, PCR and Pastorex Staph-Plus correctly identified all S. aureus SCVs, API-Staph and VITEK-2 yielded identification rates of only 40% and 10%, respectively. ConclusionsOverall, the present study has shown that commercially available PDFs induce S. aureus SCVs in vitro, which are difficult to identify and test for antimicrobial susceptibility and can potentially lead to recurrent or persistent infections. Thus, they represent a potentially underappreciated challenge not only for microbiologists, but also for clinicians.

Effect of extracellular polymeric substances on the colony size and morphological changes of Microcystis.

Surface blooms of colony-forming Microcystis are increasingly occurring in aquatic ecosystems on a global scale. Recent studies have found that the Microcystis colonial morphology is a crucial factor in the occurrence, persistence, and dominance of Microcystis blooms, yet the mechanism driving its morphological dynamics has remained unknown. This study conducted a laboratory experiment to test the effect of extracellular polymeric substances on the morphological dynamics of Microcystis. Ultrasound was used to disaggregate colonies, isolating the cells and of the Microcystis suspension. The single cells were then re-cultured under three homologous EPS concentrations: group CK, group Low, and group High. The size, morphology, and EPS [including tightly bound EPS (TB-EPS), loosely bound EPS (LB-EPS), bound polysaccharides (B-polysaccharides), and bound proteins (B-proteins)] changes of colonies were closely monitored over a period of 2 months. It was observed that colonies were rapidly formed in group CK, with median colony size (D50) reaching 183 µm on day 12. The proportion of colonies with a size of 150-500 µm increased from 1% to more than 50%. Colony formation was also observed in both groups Low and High, but their D50 increased at a slower rate and remained around 130 µm after day 17. Colonies with a size of 50-150 µm account for more than 50%. Groups CK and Low successively recovered the initial Microcystis morphology, which is a ring structure formed of several small colonies with a D50 of 130 µm. During the recovery of the colony morphology, the EPS per cell increased and then decreased, with TB-EPS and B-polysaccharides constituting the primary components. The results suggest that colony formation transitioned from adhesion driven to being division driven over time. It is suggested that the homologous EPS released into the ambient environment due to the disaggregation of the colony is a chemical cue that can affect the formation of a colony. This plays an important but largely ignored role in the dynamics of Microcystis and surface blooms.

Molecular characterization of a carbon dioxide-dependent Proteus mirabilis small-colony variant isolated from a clinical specimen

IntroductionCarbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. MethodsThe DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis, we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. ResultsWe found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. ConclusionsThe can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation.

Pollen diet diversity across bee lineages varies with lifestyle rather than colony size.

The shift to a pollen diet and the evolution of more highly organized societies, i.e., eusocial, were key milestones in bee diversification over their evolutionary history, culminating in a high dependence on feeding broods with a large variety of floral resources. Here, we hypothesized that obligatory eusocial bees have a wider diet diversity than their relatives with solitary lifestyles, and this would be related to colony size. To test both hypotheses, we surveyed diet breadth data (palynological analysis) based on the Shannon-Wiener index (H') for 85 bee taxa. We also obtained colony size for 47 eusocial bee species. These data were examined using phylogenetic comparative methods. The results support the generalist strategy as a derived trait for the bee taxa evaluated here. The dietary diversity of eusocial bees (H': 2.1, on average) was 67.5% higher than that of noneusocial bees (H': 1.21, on average). There was, however, no relationship between diet breadth and colony size, indicating that smaller colonies can harvest a pollen variety as diverse as larger colonies. Taken together, these results provide new insights into the impact of lifestyle on the diversity of collected pollen. Furthermore, this work sheds light on an advantage of living in more highly structured societies irrespective of the size of the colony.

Evaluating risks to seabirds on the urban–coastal interface: Modelling dog attacks on little penguin populations in Tasmania

Abstract Tasmania (including offshore islands) has the largest breeding population of little penguins (Eudyptula minor) in Australia, but coastal development around nesting areas has led to habitat loss, often resulting in smaller, fragmented colonies. Colonies have also been subject to predation by introduced domestic species. This study assessed whether historical levels of domestic dog predation could threaten the viability of penguin populations. Data collected since the 1980s detailing little penguin kills by domestic dogs were used to inform the extent of individual mortality events. The potential impact of dog attacks on the viability of little penguin colonies of the sizes typically found on mainland Tasmania was explored using an age‐based population model. Simulation was conducted over decadal timescales to assess the impact of attacks at varying frequencies and intensities on small (100 birds), medium (300 birds) and large (500 birds) colonies that were stable, growing or decreasing. Results suggested that frequent and intense dog attacks markedly increased the probability of colony decline, regardless of colony size. Even large, growing penguin colonies were at risk of decline. The risk of extinction was <10% for medium and large stable colonies if no more than 15–25 penguins were killed per attack and there were no more than 20 attacks per 50 years. For small‐sized colonies, the risk of extinction was <10% if fewer than 10 attacks occurred per 50‐year period and no more than 15 birds were killed per attack. Importantly, results suggested that for small colonies especially, even low levels of predator attack could lead to colony collapse in 10–15 years. For large colonies that were already decreasing, the average time to extinction was markedly increased with additional mortalities from dogs, highlighting the need for ongoing management measures that reduce or eliminate dog attacks to help conserve this iconic species.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution.

This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed. The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined. The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis. This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Evaluating Acheta domesticus (Orthoptera: Gryllidae) for the reduction of fumonisin B1 levels in livestock feed.

Mycotoxins that contaminate grain can cause the devaluation of agricultural products and create health risks for the consumer. Fumonisins are one such mycotoxin. Produced primarily by Fusarium verticillioides (Hypocreales: Nectriaceae) (Nirenberg, 1976) on corn, fumonisins' economic impact can be significant by causing various diseases in livestock if contaminated corn is not monitored and removed from animal feed. Finding safe alternatives to the destruction and waste of contaminated grain and restoring its economic value is needed for a sustainable future. Safe reintroduction into the farm food web may be possible through a consumable intermediary such as insects. This study demonstrates the suitability of the house cricket, Acheta domesticus L., as an alternative protein source in domestic animal feed by quantifying fumonisin B1 (FB1) levels in their subsequent insect meal and frass. Small colonies of 2nd instar A. domesticus were reared to 5th instar adults on nutrient-optimized corn-based diets treated with 4 levels of FB1 from 0 to 20 ppm. Increasing levels of FB1 had no adverse effects on the survivorship or growth of A. domesticus. Insect meals prepared from A. domesticus had significantly lower levels of FB1, at 3%-5% of their respective diets, while frass did not differ significantly from their diet. The successful rearing to adulthood of A. domesticus on fumonisin-contaminated diet paired with lower levels of FB1 in their processed insect meal supports the idea that more sustainable agricultural practices can be developed through remediation of low-value mycotoxin-contaminated grain with safer, higher-value insects as livestock feed components.

Selection of ethanol tolerant strains of Candida albicans by repeated ethanol exposure results in strains with reduced susceptibility to fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Metastable microstructure evolution and grain refinement in a Low-Ta containing γ-TiAl alloy through heat treatment

The paper comprehensively describes the evolution of metastable microstructures in a cast low-Ta-containing γ-TiAl alloy during cooling-tempering-lamellarization heat treatments. The behavior and mechanism of microstructure refinement are discussed. The rapid cooling generates metastable microstructures, including Widmanstätten, feathery, and massive structures, with many γ variants in small sizes and different orientations. It significantly refines the coarsened colonies and randomizes the texture. The tempering can effectively stabilize the metastable microstructures. During tempering, the γ→α phase transformation is more active at higher temperatures while coarsening and recrystallization dominate at lower temperatures. After tempering, a refined microstructure with spherical grains and convoluted structures forms. Subsequent lamellarization treatment optimizes the microstructure and obtains a fine-grained nearly-lamellar microstructure with small colonies (∼70 μm) and dispersed fine γ grains (∼10 vol%), which yielded a 1.0 % elongation with a 487 MPa yield strength (0.2%) and a 589 MPa ultimate tensile strength at room temperature. Finally, the unique promotion effects and mechanism of a small amount of Ta addition on the nucleation, growth, evolution, and stabilization of metastable microstructures are concluded and discussed, which is crucial for the refinement of cast γ-TiAl alloy by heat treatment alone.

Deep-sea ctenostome bryozoans: revision of the family Pachyzoidae, with description of a new genus and three new species from Zealandia

Pachyzoidae is a little-known family of deep-sea ctenostome Bryozoa that until now was monospecific for Pachyzoon atlanticum. Originally described from the Atlantic Ocean, the genus was also found off southeastern New Caledonia in deep waters of the geological continent of Zealandia. Pachyzoon atlanticum forms globular to flat round colonies, living on soft, muddy to sandy bottoms with a few rhizoidal cystid appendages extending from the basal, substrate-oriented side. In this study, we investigate additional pachyzoids, collected between 1965 and 2015 from over 40 sites around New Zealand, by means of detailed morphological and histological investigations. In total, several hundred colonies were encountered in the NIWA Invertebrate Collection, comprising two new species of the genus Pachyzoon, P. grischenkoi sp. nov. and P. pulvinaris sp. nov., and the new genus and species Jeanloupia zealandica gen. et sp. nov.. The genus Jeanloupia is characterized by small disc-shaped colonies with highly elongated peristomes and a quadrangular aperture, distinct from the round apertures of the genus Pachyzoon. Pachyzoid species differ in colony structure and shape, apertural papillae and polypide features such as tentacle number or digestive-tract details. Cystid appendages are non-kenozooidal, but may originate from laterally flanking kenozooids. Based on published images, alleged P. atlanticum from New Caledonia is re-interpreted as P. pulvinaris n. sp.. Morphological characters support alcyonidioidean relationships, as previously suggested. First observations on pachyzoid reproduction show macrolecithal oocytes and brooding of embryos, which seems to be the general pattern for this family. The occurrence of three new Zealandian species in a comparatively small geographical area far from the Atlantic indicates a high possibility of more species to discovered.

Differences in oxazolidinone resistance mechanisms and small colony variants emergence of Staphylococcus aureus induced in an in vitro resistance development model

ABSTRACT Invasive Staphylococcus aureus infections are associated with a high burden of disease, case fatality rate and healthcare costs. Oxazolidinones such as linezolid and tedizolid are considered potential treatment choices for conditions involving methicillin resistance or penicillin allergies. Additionally, they are being investigated as potential inhibitors of toxins in toxin-mediated diseases. In this study, linezolid and tedizolid were evaluated in an in vitro resistance development model for induction of resistance in S. aureus. Whole genome sequencing was conducted to elucidate resistance mechanisms through the identification of causal mutations. After inducing resistance to both linezolid and tedizolid, several partially novel single nucleotide variants (SNVs) were detected in the rplC gene, which encodes the 50S ribosome protein L3 in S. aureus. These SNVs were found to decrease the binding affinity, potentially serving as the underlying cause for oxazolidinone resistance. Furthermore, in opposite to linezolid we were able to induce phenotypically small colony variants of S. aureus after induction of resistance with tedizolid for the first time in literature. In summary, even if different antibiotic concentrations were required and SNVs were detected, the principal capacity of S. aureus to develop resistance to oxazolidinones seems to differ between linezolid and tedizolid in-vivo but not in vitro. Stepwise induction of resistance seems to be a time and cost-effective tool for assessing resistance evolution. Inducted-resistant strains should be examined and documented for epidemiological reasons, if MICs start to rise or oxazolidinone-resistant S. aureus outbreaks become more frequent.

An Integrated Undergraduate Laboratory Exercise to Demonstrate Microbial Evolution

Understanding that evolution progresses through generation of DNA variants followed by selection is a key learning outcome for biology students. We designed an integrated and innovative undergraduate laboratory exercise using Saccharomyces cerevisiae to demonstrate these principles. Students perform in vitro experimental evolution by repeatedly propagating large or small yeast colonies on a weekly basis. Small-colony variants known as petites arise by mutations that disrupt aerobic respiration. To demonstrate the effects of increased mutation rates, half of the selection lines are exposed to ultraviolet irradiation. To understand how the petite phenotype arises, polymerase chain reaction (PCR) is performed to examine mitochondrial DNA, while biochemical assays are used to assess the ability of petites to undergo aerobic respiration. This exercise demonstrates evolution by artificial selection over a suitably short timeframe and links the results to a critical biochemical process: the role of mitochondria in aerobic respiration and ATP production. By implementing these experiments, we successfully demonstrated that the frequencies of petite mutants in evolved populations varied according to the selection pressure we applied, and that petite mutants carried deletions in mitochondrial DNA as anticipated. Through an integrated learning context, this practical exercise promotes fundamental understanding of evolutionary processes and fosters critical thinking skills.

Demographic recovery of corals at a wave-exposed reef following catastrophic disturbance

Rapid recovery of coral cover following acute disturbance has been documented on many reefs. Yet measuring coverage alone can mask shifts in community and demographic structure. Here, we quantify long-term changes in population size structure for three common genera (Acropora, Pocillopora, and Stylophora) at an eastern outer reef in Palau, Micronesia, following catastrophic loss of corals due to typhoon Bopha in 2012, based on size measurements from 3648 coral colonies. Mean colony size returned to pre-disturbance levels within 4 and 6 years for Stylophora and Pocillopora, respectively. By 2020, Pocillopora colony density far exceeded pre-disturbance levels, with rapid successful recruitment following typhoon Bopha. Despite recovery of Acropora colony density by 2020, populations remained dominated by smaller colonies. We demonstrate that full demographic recovery can occur more rapidly for pocilloporids (within 6 years) compared to Acropora which had not fully recovered by 8 years post-disturbance, possibly due to fewer annual recruitment events and larger maximum colony sizes. Our results highlight the value of demographic metrics as early indicators of recovery.

Mode of Antibacterial Action of Tomatidine C3-Diastereoisomers.

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the Staphylococcus aureus small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-β) and TM-03 (C3-α). These molecules are equally potent against prototypical S. aureus and E. coli strains (MIC 8 and 32 µg/mL, respectively), whereas TM-02 is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable E. coli strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited E. coli ATP synthase, with Ki values of 1.1 µM and 3.5 µM for TM-02 and TM-03, respectively, and the bactericidal effect of TM-02 was linked to ATP synthase inhibition. Furthermore, TM-02 had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, TM-03 caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α.