Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium.

BACKGROUND: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. METHODS: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. RESULTS: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. CONCLUSIONS: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.

Propranolol Reduces the Development of Lesions and Rescues Barrier Function in Cerebral Cavernous Malformations: A Preclinical Study.

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Nail Gun Suicide: An Atypical Case Report and Review of the Literature.

ABSTRACT: Nail guns are pneumatically driven or powder-actuated tools, which are used to drive nails into wood or concrete. A 42-year-old man, who was a builder by profession and history of antidepressant use, was found dead in his vehicle, holding a "Ramset" nail gun in his right hand. A straight metal nail and a book with a small hole were recovered from the scene.At autopsy, an irregularly circular puncture wound was seen on the forehead and a cruciate laceration was seen on the posterior aspect of the scalp. No burning, blackening, or tattooing was present around either injury. Both internal and external beveling was present with the latter being relatively larger. Toxicological analysis revealed alcohol and methamphetamine in blood. Psychiatric history and substance abuse must also be considered when individuals in construction industry are given access to tools like nail guns.Several unique features not previously reported were observed in this case. The deceased had overcome the built-in safety mechanism of the nail gun, by placing a book in between his forehead and muzzle. Another unique feature is that the nail had exited the cranium. Beveling, a feature commonly found in firearm injuries, was also seen in this case.

Survey on postmortem screening and management of COVID-19 related deaths.

The COVID-19 pandemic is associated with a high case fatality rate in some countries even thought the majority of cases are asymptomatic. Scientific studies on this novel virus is limited and there is uncertainty regarding the best practices for death investigations both in terms of detection of the disease as well as autopsy safety. An online survey was conducted to identify how different institutions responded to the screening and management of dead bodies during the early phase of the pandemic from January to May. A questionnaire was developed using Google Forms and data was collected from 14 different forensic and pathological institutions in 9 countries. None of the institutions had performed any screening prior to March. Four institutions stated that screening was done routinely. In total, 322 cases had been screened using RT-PCR, out of which 40 positive cases were detected among four institutions. The commonest types of samples obtained were nasopharyngeal and oropharyngeal swabs which also had the highest rates of positivity followed by tracheal swab. Blood, swabs from cut surfaces of lung and lung tissue also gave positive results in some cases. Majority of the positive cases were > 65 years with a history suggestive of respiratory infection and were clinically suspected to have COVID-19 before death. Except for one institution which performed limited dissections, standard autopsies were conducted on all positive cases. Disposal of bodies involved the use of sealed body bags and labelling as COVID positive. Funeral rites were restricted and none of the institutions advocated cremation. There were no reports of disease transmission to those who handled COVID positive bodies.

Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii.

The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.IMPORTANCEAcinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

Effect of Incubation Temperature on Antibiotic Resistance and Virulence Factors of Acinetobacter baumannii ATCC 17978.

Acinetobacter baumannii is a notorious opportunistic pathogen that is prevalent mainly in hospital settings. The ability of A. baumannii to adapt and to survive in a range of environments has been a key factor for its persistence and success as an opportunistic pathogen. In this study, we investigated the effect of temperature on the clinically relevant phenotypes displayed by A. baumannii at 37°C and 28°C. Surface-associated motility was significantly reduced at 28°C, while biofilm formation on plastic surfaces was increased at 28°C. Decreased susceptibility to aztreonam and increased susceptibility to trimethoprim-sulfamethoxazole were observed at 28°C. No differences in virulence, as assayed in a Galleria mellonella model, were observed. Proteomic analysis showed differential expression of 629 proteins, of which 366 were upregulated and 263 were downregulated at 28°C. Upregulation of the Csu and iron uptake proteins at 28°C was a key finding for understanding some of the phenotypes displayed by A. baumannii at 28°C.

New ß-lactam - Tetramic acid hybrids show promising antibacterial activities.

ß-Lactams are the most important class of antibiotics, for which the emergence of resistance threatens their utility. As such, we explored the extent to which the tetramic acid motif, frequently found in naturally occurring antibiotics, can be used to generate novel ß-lactam antibiotics with improved antibacterial activity. We synthesized new ampicillin - tetramic acid, cephalosporin - tetramic acid, and cephamycin - tetramic acid analogs and evaluated their activities against problematic Gram-positive and Gram-negative pathogens. Amongst the analogs, a 7-aminocephalosporanic acid analog, 3397, and a 7-amino-3-vinyl cephalosporanic acid, 3436, showed potent activities against S. aureus NRS 70 (MRSA) with MICs of 6.25 µg/mL and 3.13 µg/mL respectively. These new analogs were ≥16-fold more potent than cefaclor and cephalexin. Additionally, a Δ2 cephamycin - tetramic acid analog 3474 which contained a basic guanidinium substituent at the 5-position of the tetramic acid core displayed potent activity against several clinical strains of K. pneumoniae and E. coli.

Prevalence of antibiotic resistance genes in bacterial communities associated with Cladophora glomerata mats along the nearshore of Lake Ontario.

The alga Cladophora glomerata can erupt in nuisance blooms throughout the lower Great Lakes. Since bacterial abundance increases with the emergence and decay of Cladophora, we investigated the prevalence of antibiotic resistance (ABR) in Cladophora-associated bacterial communities up-gradient and down-gradient from a large sewage treatment plant (STP) on Lake Ontario. Although STPs are well-known sources of ABR, we also expected detectable ABR from up-gradient wetland communities, since they receive surface run-off from urban and agricultural sources. Statistically significant differences in aquatic bacterial abundance and ABR were found between down-gradient beach samples and up-gradient coastal wetland samples (ANOVA, Holm-Sidak test, p < 0.05). Decaying and free-floating Cladophora sampled near the STP had the highest bacterial densities overall, including on ampicillin- and vancomycin-treated plates. However, quantitative polymerase chain reaction analysis of the ABR genes ampC, tetA, tetB, and vanA from environmental communities showed a different pattern. Some of the highest ABR gene levels occurred at the 2 coastal wetland sites (vanA). Overall, bacterial ABR profiles from environmental samples were distinguishable between living and decaying Cladophora, inferring that Cladophora may control bacterial ABR depending on its life-cycle stage. Our results also show how spatially and temporally dynamic ABR is in nearshore aquatic bacteria, which warrants further research.

Synthesis and Evaluation of Thiazolidine Amide and N-Thiazolyl Amide Fluoroquinolone Derivatives.

In an effort to develop new fluoroquinolones, we synthesized eight compounds and tested them against a panel of bacteria. The design of these compounds was guided by the introduction of the isothiazoloquinolone motif. The three most active compounds in this series, 8-10, demonstrated good antibacterial activity against methicillin-sensitive Staphylococcus aureus and healthcare-acquired methicillin-resistant Staphylococcus aureus (MIC 0.62-6.3 µg/mL). Further, when these three active compounds were tested for their inhibitory effects on bacterial enzymes, compound 9 was the most effective agent exhibiting IC50 values of 33.9 and 116.5 µM in the S. aureus deoxyribonucleic acid (DNA) gyrase supercoiling and topoisomerase IV decatenation assays, respectively.

Characteristics of road traffic accident casualties admitted to a tertiary care hospital in Sri Lanka.

The mortality and morbidity of road traffic accidents (RTA) is increasing in the South Asian region, including Sri Lanka. Therefore, the demographic factors, types of vehicles involved, and the severity of injuries sustained in RTA was studied. Age, gender, and details of the incident of all patients admitted to hospital following a RTA, between January 2007 and August 2012, were obtained by interview. Following a medico-legal examination, the type and severity of injuries was categorized as, non-grievous, grievous, endangering life or fatal in the ordinary course of nature. Of the 579 RTA casualties examined, 72% were males, 28% females, and 26% were in the 20-29 year age group. There were 44% passengers, 32% drivers, and 20% pedestrians. Of the 440 vehicle occupants, 37% were on motor cycles, 28% in three wheelers, 13% in dual purpose vehicles and 11% in buses. Of the 114 pedestrians, 33% had been struck by motor cycles, 19% by three-wheelers and 17% by dual purpose vehicles. There was at least one soft tissue injury in 84%, whilst 45% had one or more fractures. In 85% of bicycle riders, the injuries were grievous, endangering life or fatal in the ordinary course of nature. A high proportion of young adults sustained grievous injuries due to RTA. Almost two thirds of the casualties resulted from motorcycle or three wheeler accidents. Laws limiting the number of passengers carried, installation of side doors, mandatory use of seat belts in three wheelers, and protective garments for motorcyclists are recommended.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada.

UNLABELLED: Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, ß-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE: In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.

Adjuvants Based on Hybrid Antibiotics Overcome Resistance in Pseudomonas aeruginosa and Enhance Fluoroquinolone Efficacy.

The use of adjuvants that rescue antibiotics against multidrug-resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of ß-lactam antibiotics and ß-lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram-negative pathogens has been challenging. We describe tobramycin-ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug-resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure-activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.

Penetrative Injury to the Face Resulting in Delayed Death After Rupture of a Cavernous Sinus Aneurysm on the Contralateral Side.

Delayed deaths after assault give rise to medicolegal issues such as whether the assault was directly responsible, did it contribute or was it totally unrelated to the death. We present the case of a patient who sustained penetrative trauma to the face resulting in the formation of a contralateral, carotid-cavernous fistula, which resulted in aneurysm formation, delayed rupture, and death. A literature search failed to find a similar case.

Triclosan can select for an AdeIJK-overexpressing mutant of Acinetobacter baumannii ATCC 17978 that displays reduced susceptibility to multiple antibiotics.

In order to determine if triclosan can select for mutants of Acinetobacter baumannii ATCC 17978 that display reduced susceptibilities to antibiotics, we isolated a triclosan-resistant mutant, A. baumannii AB042, by serial passaging of A. baumannii ATCC 17978 in growth medium supplemented with triclosan. The antimicrobial susceptibility of AB042 was analyzed by the 2-fold serial dilution method. Expression of five different resistance-nodulation-division (RND) pump-encoding genes (adeB, adeG, adeJ, A1S_2818, and A1S_3217), two outer membrane porin-encoding genes (carO and oprD), and the MATE family pump-encoding gene abeM was analyzed using quantitative reverse transcriptase (qRT) PCR. A. baumannii AB042 exhibited elevated resistance to multiple antibiotics, including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline, and trimethoprim-sulfamethoxazole, in addition to triclosan. Genome sequencing of A. baumannii AB042 revealed a (116)G→V mutation in fabI, the gene encoding the target enzyme for triclosan. Expression analysis of efflux pumps showed overexpression of the AdeIJK pump, and sequencing of adeN, the gene that encodes the repressor of the adeIJK operon, revealed a 73-bp deletion which would cause a premature termination of translation, resulting in an inactive truncated AdeN protein. This work shows that triclosan can select for mutants of A. baumannii that display reduced susceptibilities to multiple antibiotics from chemically distinct classes in addition to triclosan resistance. This multidrug resistance can be explained by the overexpression of the AdeIJK efflux pump.

Necrotizing fasciitis and death following an insect bite.

A healthy man who presented to hospital with painful swelling of the left arm following a history of insect (tick) bite developed shock and died within 48 hours. The postmortem examination revealed swelling and desquamation of skin and erythema on the left arm extending below the elbow. The subcutaneous tissue was necrotic with healthy underlying muscles. Group A ß-hemolytic streptococcus was isolated from postmortem swabs of the infected tissue. Histopathologic changes were consistent with necrotizing fasciitis.Secondary bacterial infection is an important possible complication following insect bite, and a high degree of suspicion with aggressive early treatment is required in cases of necrotizing fasciitis to prevent fatalities. Both clinicians and pathologists need to be aware of this rare, rapidly fatal condition that may follow an insect bite.

Antibiotic resistance and expression of resistance-nodulation-division pump- and outer membrane porin-encoding genes in Acinetobacter species isolated from Canadian hospitals.

BACKGROUND: Bacterial pathogens belonging to the genus Acinetobacter cause serious infections in immunocompromised individuals that are very difficult to treat due to their extremely high resistance to many antibiotics. OBJECTIVE: To investigate the role of resistance-nodulation-division (RND) pumps and porins in the antibiotic resistance of Acinetobacter species collected from Canadian hospitals. METHODS: Clinical isolates of Acinetobacter species collected from Canadian hospitals were analyzed for the expression of genes encoding RND pumps (adeB, adeG, adeJ, AciBau_2746 and AciBau_2436) and outer membrane porins (carO, 33 kDa porin and oprD) using quantitative reverse transcription (qRT) polymerase chain reaction. Species identification of the isolates was performed using a multiplex polymerase chain reaction method for gyrB. RESULTS: The expression of RND pump-encoding genes was widespread in the clinical isolates of Acinetobacter species, with each of the isolates expressing at least one RND pump. adeG was found to be overexpressed in all of the isolates, while adeB was found to be overexpressed in only two isolates. Among the porin-encoding genes, the expression of carO was considerably downregulated among the majority of isolates. CONCLUSION: The present study was the first to analyze the expression of RND pump- and porin-encoding genes in the clinical isolates of Acinetobacter species from Canadian hospitals. The overexpression of genes encoding RND pumps and the downregulation of genes encoding porins was common in clinical isolates of Acinetobacter species from Canadian hospitals, with the AdeFGH pump being the most commonly expressed RND pump.

HISTORIQUE: Les pathogènes bactériens qui appartiennent au gène Acinetobacter provoquent de graves infections chez les personnes immunocompromises. Ces infections sont très difficiles à traiter en raison de leur résistance extrêmement élevée à de nombreux antibiotiques. OBJECTIF: Examiner le rôle des pompes RND (resistance-nodulation-division) et des porines dans l'antibiorésistance des espèces d'Acinetobacter prélevées dans des hôpitaux canadiens. MÉTHODOLOGIE: Les chercheurs ont analysé des isolats cliniques d'espèces d'Acinetobacter prélevés dans des hôpitaux canadiens afin de déceler l'expression des gènes codants des pompes RND (adeB, adeG, adeJ, AciBau_2746 et AciBau_2436) et des porines de la membrane externe (carO, porine 33 kDa et oprD) à l'aide de la réaction en chaîne de la polymérase après transcription inverse quantitative (qRT). Ils ont identifié les espèces d'isolats à l'aide d'une méthode de réaction en chaîne de la polymérase multiplexe pour le gyrB. RÉSULTATS: L'expression de gènes codants de la pompe RND était généralisée dans les isolats cliniques des espèces d'Acinetobacter, chaque isolat exprimant au moins une pompe RND. Les chercheurs ont observé une surexpression de l'adeG dans tous les isolats, mais la surexpression de l'adeB dans seulement deux isolats. Parmi les gènes codants des porines, l'expression du carO présentait une régulation négative marquée dans la majorité des isolats. CONCLUSION: La présente étude était la première à analyser l'expression des gènes codants de la pompe d'efflux RND et des porines dans les isolats cliniques d'espèces d'Acinetobacter provenant d'hôpitaux canadiens. La surexpression des gènes codants des pompes RND et la régulation négative des gènes codants des porines était courante dans les isolats cliniques d'espèces d'Acinetobacter provenant d'hôpitaux canadiens, la pompe AdeFGH étant la pompe d'efflux RND la plus exprimée.

Electron tomography unravels new insights into fiber cell wall nanostructure; exploring 3D macromolecular biopolymeric nano-architecture of spruce fiber secondary walls.

Lignocellulose biomass has a tremendous potential as renewable biomaterials for fostering the "bio-based society" and circular bioeconomy paradigm. It requires efficient use and breakdown of fiber cell walls containing mainly cellulose, hemicellulose and lignin biopolymers. Despite their great importance, there is an extensive debate on the true structure of fiber walls and knowledge on the macromolecular nano-organization is limited and remains elusive in 3D. We employed dual-axis electron tomography that allows visualization of previously unseen 3D macromolecular organization/biopolymeric nano-architecture of the secondary S2 layer of Norway spruce fiber wall. Unprecedented 3D nano-structural details with novel insights into cellulose microfibrils (~ 2 nm diameter), macrofibrils, nano-pore network and cell wall chemistry (volume %) across the S2 were explored and quantified including simulation of structure related permeability. Matrix polymer association with cellulose varied between microfibrils and macrofibrils with lignin directly associated with MFs. Simulated bio-nano-mechanical properties revealed stress distribution within the S2 and showed similar properties between the idealized 3D model and the native S2 (actual tomogram). Present work has great potential for significant advancements in lignocellulose research on nano-scale understanding of cell wall assembly/disassembly processes leading to more efficient industrial processes of functionalization, valorization and target modification technologies.

Growth phase-dependent expression of RND efflux pump- and outer membrane porin-encoding genes in Acinetobacter baumannii ATCC 19606.

OBJECTIVES: To analyse the expression of resistance-nodulation-division (RND) efflux pumps and outer membrane porins during various growth phases of Acinetobacter baumannii ATCC 19606. METHODS: Expression of five different RND pump-encoding genes (adeB, adeG, adeJ, AciBau_2436 and AciBau_2746) and two outer membrane porin-encoding genes (carO and oprD) was analysed at four growth timepoints, representing early, mid and late log phases and the stationary phase using quantitative RT-PCR. RESULTS: The adeB and adeJ RND pump genes were expressed at higher levels than all of the other RND pumps, though their expression was reduced at higher cell density. The adeG gene was found to be constitutively expressed, albeit at much lower levels than those seen for adeB or adeJ. The previously uncharacterized AciBau_2436 was found to be expressed in the early exponential phase. The expression level of oprD was found to be inversely proportional to cell density, while that of carO was found to increase in the mid-log phase but then decrease in the later stages of the growth phase. CONCLUSIONS: This work shows cell density-dependent expression of adeB, adeJ, AciBau_2436, carO and oprD genes, suggesting a role of global regulatory mechanisms in the expression of these genes in A. baumannii ATCC 19606.