Third generation cephalosporin-resistant Klebsiella pneumoniae thriving in patients and in wastewater: what do they have in common?

BACKGROUND: Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3rd generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses. RESULTS: Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin. CONCLUSIONS: It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.

Food Ingredients Derived from Lemongrass Byproduct Hydrodistillation: Essential Oil, Hydrolate, and Decoction.

Essential oil (EO), hydrolate, and nondistilled aqueous phase (decoction) obtained from the hydrodistillation of lemongrass byproducts were studied in terms of their potential as food ingredients under a circular economy. The EO (0.21%, dry weight basis) was composed mainly of monoterpenoids (61%), the majority being citral (1.09 g/kg). The minimal inhibitory concentrations (MIC) of lemongrass EO against Escherichia coli, Salmonella enterica, and Staphylococcus aureus, were 617, 1550, and 250 µg/mL, respectively. This effect was dependent on the citral content. Particularly for Gram-negative bacteria, a synergism between citral and the remaining EO compounds enhanced the antimicrobial activity. The polymeric material obtained from the nondistilled aqueous phase was composed of phenolic compounds (25% gallic acid equivalents) and carbohydrates (22%), mainly glucose (66 mol%). This polymeric material showed high antioxidant activity due to bound phenolic compounds, allowing its application as a functional dietary fiber ingredient. Matcha green tea formulations were successfully mixed with lemongrass hydrolate containing 0.21% EO (dry weight basis) with 58% of monoterpenoids, being citral at 0.73 g/kg, minimizing matcha astringency with a citrus flavor and extending the product shelf life. This holistic approach to essential oils' hydrodistillation of Cymbopogon citratus byproducts allows for valorizing of the essential oil, hydrolate, and decoction for use as food ingredients.

Stroke Care during the COVID-19 Pandemic: International Expert Panel Review.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has placed a tremendous strain on healthcare services. This study, prepared by a large international panel of stroke experts, assesses the rapidly growing research and personal experience with COVID-19 stroke and offers recommendations for stroke management in this challenging new setting: modifications needed for prehospital emergency rescue and hyperacute care; inpatient intensive or stroke units; posthospitalization rehabilitation; follow-up including at-risk family and community; and multispecialty departmental developments in the allied professions. SUMMARY: The severe acute respiratory syndrome coronavirus 2 uses spike proteins binding to tissue angiotensin-converting enzyme (ACE)-2 receptors, most often through the respiratory system by virus inhalation and thence to other susceptible organ systems, leading to COVID-19. Clinicians facing the many etiologies for stroke have been sobered by the unusual incidence of combined etiologies and presentations, prominent among them are vasculitis, cardiomyopathy, hypercoagulable state, and endothelial dysfunction. International standards of acute stroke management remain in force, but COVID-19 adds the burdens of personal protections for the patient, rescue, and hospital staff and for some even into the postdischarge phase. For pending COVID-19 determination and also for those shown to be COVID-19 affected, strict infection control is needed at all times to reduce spread of infection and to protect healthcare staff, using the wealth of well-described methods. For COVID-19 patients with stroke, thrombolysis and thrombectomy should be continued, and the usual early management of hypertension applies, save that recent work suggests continuing ACE inhibitors and ARBs. Prothrombotic states, some acute and severe, encourage prophylactic LMWH unless bleeding risk is high. COVID-19-related cardiomyopathy adds risk of cardioembolic stroke, where heparin or warfarin may be preferable, with experience accumulating with DOACs. As ever, arteritis can prove a difficult diagnosis, especially if not obvious on the acute angiogram done for clot extraction. This field is under rapid development and may generate management recommendations which are as yet unsettled, even undiscovered. Beyond the acute management phase, COVID-19-related stroke also forces rehabilitation services to use protective precautions. As with all stroke patients, health workers should be aware of symptoms of depression, anxiety, insomnia, and/or distress developing in their patients and caregivers. Postdischarge outpatient care currently includes continued secondary prevention measures. Although hoping a COVID-19 stroke patient can be considered cured of the virus, those concerned for contact safety can take comfort in the increasing use of telemedicine, which is itself a growing source of patient-physician contacts. Many online resources are available to patients and physicians. Like prior challenges, stroke care teams will also overcome this one. Key Messages: Evidence-based stroke management should continue to be provided throughout the patient care journey, while strict infection control measures are enforced.

Gut and faecal bacterial community of the terrestrial isopod Porcellionides pruinosus: potential use for monitoring exposure scenarios.

This work aimed to characterize the gut and faeces bacterial communities (BC) of Porcellionides pruinosus using high-throughput sequencing. Isopods were collected from the field and kept in laboratory conditions similar to those normally applied in ecotoxicology tests. Faeces and purged guts of isopods (n = 3 × 30) were analysed by pyrosequencing the V3-V4 region of 16 S rRNA encoding gene. Results showed that gut and faecal BCs were dominated by Proteobacteria, particularly by an OTU (Operational Taxonomic Unit) affiliated to genus Coxiella. Diversity and richness values were statistically higher for faecal BC, mainly due to the occurrence of several low-abundance phylotypes. These results may reflect faecal carriage of bacterial groups that cannot settle in the gut. BCs of P. pruinosus comprised: (1) common members of the soil microbiota, (2) bacterial symbionts, (3) bacteria related to host metabolic/ecological features, and (4) bacterial etiological agents. Comparison of BC of this isopod species with the BC from other invertebrates revealed common bacterial groups across taxa. The baseline information provided by this work will assist the design and data interpretation of future ecotoxicological or biomonitoring assays where the analysis of P. pruinosus BC should be included as an additional indicator. CAPSULE: Terrestrial isopods bacterial communities might support ecotoxicological assays and biomonitoring processes as a valuable tool.

Effects of Long-Term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia toletana.

Amphibian's skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work assessed whether an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (with 18 g/L of NaCl): (1) long-term exposure (for 46 days; Et-NaCl), and (2) long-term exposure followed by a recovery period (exposure for 30 days to NaCl and then to LB medium for 16 days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by exposing them to 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). The results showed that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5 g/L (8.64-36.4) for Et-LB; 30.3 g/L (23.2-37.4) for Et-NaCl; and 26.1 g/L (19.332.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and between Et-NaCl and Et-R, suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. These findings may be further explored to be used in bioaugmentation procedures through the supplementation with this bacterium of the skin microbiome of natural populations of amphibians exposed to salinization.

Genomic analysis of Chromobacterium haemolyticum: insights into the species resistome, virulence determinants and genome plasticity.

The increasing number of Chromobacterium haemolyticum human infection reports, especially in tropical regions and connected with environmental sources, resulted in an urge to better describe this species. This study aimed to characterize the C. haemolyticum resistome, virulence determinants and genetic platforms related with genome plasticity. A comparative genomic analysis was conducted between clinical C. haemolyticum genomes publicly available and the genome of an environmental isolate obtained in this study. The pangenome of C. haemolyticum was calculated and a total of 3378 core genes were predicted in its core genome, corresponding to 51.7% of the pangenome. Genetic determinants putatively encoding resistance to beta-lactams, fosfomycin, aminoglycosides and trimethoprim were predicted in all genomes, possibly constituting the intrinsic resistome of this species. In terms of resistance to beta-lactams, 4 genes were predicted encoding beta-lactamases of classes A, C and D. Moreover, the analysis of Chromobacterium genomes and C. haemolyticum environmental isolates reinforced the role of this genus as progenitor of the blaKPC gene. Putative virulence factors (VFs) were predicted in all genomes, related to adherence, toxins production, colonization and cell invasion. Secretion systems, including type III, were detected. A significant number of transposases and genomic islands were predicted in C. haemolyticum, in some cases above the average reported for Gram-negative bacterial genomes. We conclude that C. haemolyticum strains, including those of environmental origin, present a noteworthy collection of antibiotic resistance genes and VFs. Furthermore, sequences related to gene mobility and genome plasticity suggest high adaptability potential and a possible role as disseminator of antibiotic resistance.

Inorganic nitrate prevents the loss of tight junction proteins and modulates inflammatory events induced by broad-spectrum antibiotics: A role for intestinal microbiota?

Upon consumption, dietary nitrate is reduced to nitrite in the oral cavity and to nitric oxide (•NO) in the stomach. Here, •NO increases mucosal blood flow, mucus thickness and prevents microbial infections. However, the impact of nitrate on gut microbiota, a pleiotropic organism essential to maintain gastrointestinal and systemic welfare, remains elusive. This study investigates the impact of nitrate on gut microbiota profile and ensued mucosal effects during dysbiosis. Male Wistar rats were randomly distributed in 4 groups and the drinking water was supplemented for 7 days as follows: 1) antibiotic cocktail (neomycin, bacitracin and imipenem), 2) antibiotic cocktail + sodium nitrate, 3) sodium nitrate and 4) regular drinking water. Animals were weighted daily and feces were collected before and after the treatment. The stomach was isolated and the expression of occludin, claudin-5 as well as myeloperoxidase and iNOS was studied. Bacterial DNA was analyzed in fecal samples by PCR-DGGE genetic fingerprinting. Nitrate prevented antibiotic-induced body weight loss (1.9 ± 1.8% vs 8.9 ±â€¯1.8%, p < 0.05) and cecamegalia (7.1 ±â€¯0.5% vs 5.6 ±â€¯0.4%, p < 0.05). Gastric expression of occludin and claudin-5 tended to decrease during dysbiosis but both protein levels were recovered following nitrate consumption (p < 0.05). Similarly, nitrate inhibited the overexpression of myeloperoxidase and iNOS observed under dysbiosis (p < 0.05). Broad spectrum antibiotics significantly decreased microbiota richness and diversity in comparison to controls (p = 0.0016). After 7 days of treatment, whereas antibiotics reduced microbiota richness by 56%, it was observed that nitrate was able to prevent such microbial loss to only 48%, although without statistical differences (p = 0.068). This data suggests that dietary nitrate may be envisaged as a key component of functional foods with beneficial impact on gastric mucosal integrity during antibiotherapy but further studies are mandatory to better ascertain as to whether it modulates intestinal microbiota in terms of taxonomic and functional levels.

The endosphere of the salt marsh plant Halimione portulacoides is a diversity hotspot for the genus Salinicola: description of five novel species Salinicola halimionae sp. nov., Salinicola aestuarinus sp. nov., Salinicola endophyticus sp. nov., Salinicola halophyticus sp. nov. and Salinicola lusitanus sp. nov.

Seven endophytic strains were isolated from the halophyte Halimione portulacoides, collected from Ria de Aveiro, Portugal. To determine their exact taxonomic position, comparative analyses were performed with these strains and closely related type strains of Salinicola species. Genome sequencing and comparison indicated that five of the seven isolated strains comprised distinct and novel species (average nucleotide identity <0.95; in silico DNA-DNA hybridization <70 %; G+C difference >1 %). Multilocus sequence analysis was performed using gyrB, rpoD and 16S rRNA gene sequences from the novel and type strains to determine their phylogenetic positions. The novel strains are facultative anaerobes, mesophilic, facultative alkaliphic and halophilic, test positive for catalase and oxidase activities, for hydrolysis of Tween 20 and phosphate, for production of indole-3-acetic acid, but do not produce H2S. Ubiquinone UQ-9 is present in major amounts in all strains. The major fatty acids include C16 : 0 and the summed feature containing C18 : 1ω7c and/or C18 : 1ω6c. The DNA G+C content ranges from 60.6 to 65.8 mol%. Five strains were confirmed as new species belonging to the genus Salinicola, for which the names Salinicolahalimionae sp. nov. (type strain CPA60T=CECT 9338T=LMG 30107T), Salinicolaaestuarinus sp. nov. (type strain CPA62T=CECT 9339T=LMG 30108T), Salinicolaendophyticus sp. nov. (type strain CPA92T=CECT 9340T=LMG 30109T), Salinicolahalophyticus sp. nov. (type strain CR45T=CECT 9341T=LMG 30105T) and Salinicola lusitanus sp. nov. (type strain CR50T=CECT 9342T=LMG 30106T) are proposed.

The impact of antibiotic exposure in water and zebrafish gut microbiomes: A 16S rRNA gene-based metagenomic analysis.

In order to supply human demand for food, the aquaculture industry has been growing fast in the last years, being fish usually cultivated in overcrowded conditions. Hence, to prevent the rapidly disease spreading, antibiotics may be applied to both sick and healthy animals. Due to its broad spectrum, oxytetracycline (OTC) is one of the most used antibiotics in food-production. Yet, although useful to prevent infections, antibiotics may reshape aquatic animals' microbiome, disturbing hosts' welfare. However, the impact of this exposure to the organism microbiome and its surrounding environment is poorly understood. Then, the objective of this study was to analyze in detail the long-term effect of OTC in both zebrafish gut and water microbiomes. Zebrafish adults were exposed, via water, for two months to three concentrations of OTC (0, 10 and 10000 µg/L). Total DNA was extracted from gut and water samples and the V3-V4 region of the bacterial 16 S rRNA gene was sequenced using Illumina technology. Results of alpha and beta-diversity analyses revealed that long-term exposure to OTC impacted both zebrafish gut and water microbiomes. In water samples, effects were observed even at the lowest (10 µg/L) OTC concentration tested resulting in an increase in Deltaproteobacteria, namely the Myxococcales and Bdellovibrionales orders. On the other hand, effects on zebrafish gut were only observed at the highest concentration with the selection of Alphaproteobacteria and Actinobacteria classes. Although these classes are common in fish gut, the increase of Actinobacteria may represent a health problem since some genera like Gordonia are linked to some human infection disease. Nevertheless, in both gut and water, it was observed a decrease in Gamaproteobacteria, probably due to OTC mode of action. In silico functional metagenomic analysis revealed that OTC exposure selected general detoxification mechanisms. In addition, the abundance of functional genes involved in Quorum Sensing (QS) increased under OTC exposure suggesting that QS may help bacteria to survive OTC stress. Thus, future studies should consider post-exposure scenarios for a deeper analysis of the water and zebrafish gut resistome, since bacteria may react differently after exposure ceased.

Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticals.

Pharmaceuticals are micropollutants often present in wastewater treatment systems. In this study, the potential impact of such micropollutants on the bacterial population within aerobic granular sludge (AGS) bioreactor was investigated. The AGS bacterial community structure and composition were accessed combining DGGE fingerprinting and barcoded pyrosequencing analysis. Both revealed the existence of a dynamic bacterial community, independently of the pharmaceuticals presence. The AGS microbiome at both phylum and class levels varied over time and, after stopping pharmaceuticals feeding, the bacterial community did not return to its initial composition. Nevertheless, most of the assigned OTUs were present throughout the different operational phases. This core microbiome, represented by over 72% of the total sequences in each phase, probably played an important role in biological removal processes, avoiding their failure during the disturbance period. Quantitative-PCR revealed that pharmaceuticals load led to gradual changes on the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and polyphosphate-accumulating organisms (PAO) but their persistence during that phase demonstrated the resilience of such bacterial groups. AGS microbiome changed over time but a core community was maintained, probably ensuring the accomplishment of the main biological removal processes.

mcr-1 and blaKPC-3 in Escherichia coli Sequence Type 744 after Meropenem and Colistin Therapy, Portugal.

Escherichia coli Ec36 was recovered from a patient in Portugal after treatment with meropenem and colistin. Besides an IncF plasmid with Tn1441d-blaKPC-3, already reported in clinical strains in this country, E. coli Ec36 co-harbored an IncX4::mcr-1 gene. Results highlight emerging co-resistance to carbapenems and polymyxins after therapy with drugs from both classes.

Zunongwangia endophytica sp. nov., an endophyte isolated from the salt marsh plant, Halimione portulacoides, and emended description of the genus Zunongwangia.

Taxonomical analyses were performed on strain CPA58T, a novel isolate obtained from surface-sterilized aboveground tissues of the halophyte Halimione portulacoides, collected from a salt marsh in Ria de Aveiro, Portugal. The strain was Gram-stain-negative, rod-shaped, oxidase-negative and catalase-positive. Optimal growth was observed at 26 °C, at pH 6-8 and in the presence of 2 to 3 % (w/v) NaCl. Phylogenetic analyses, based on the 16S rRNA gene sequence, showed that strain CPA58T belongs to the genus Zunongwangia, with highest sequence similarities to both Zunongwangia profunda SM-A87T and Zunongwangia mangrovi P2E16T (96.5 %), followed by Zunongwangia atlantica 22II14-10F7T (95.9 %). The principal fatty acids were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and iso-C17 : 0 3-OH. The major respiratory quinone was MK-6 and the DNA G+C content was 35.1 mol%. Phylogenetic and chemotaxonomic analyses clearly placed strain CPA58T in the genus Zunongwangia. However, 16S rRNA gene sequence analysis showed that the threshold for same species relatedness was not surpassed, and biochemical tests revealed diagnostic characteristics that differentiated this strain from other type strains of species of the genus Zunongwangia. Overall, the analyses showed that strain CPA58T represents a novel species within the genus Zunongwangia, for which the name Zunongwangia endophytica sp. nov. is proposed, with the type strain CPA58T (=CECT 9128T=LMG 29517T).

Saccharospirillum correiae sp. nov., an endophytic bacterium isolated from the halophyte Halimione portulacoides.

A Gram-stain negative, oxidase- and catalase- positive, motile, aerobic, non-pigmented spirillum, designated CPA1T, was isolated from the surface-sterilized tissues of a halophyte, Halimione portulacoides, collected from a salt marsh in Aveiro, Portugal. The isolate was mesophilic, facultatively alkaliphilic and halophilic, and grew between 18 and 42.5 °C (optimum 30 °C), from pH 5.0 to 11.5 (optimum 7.0-7.5), from 0.5 to 5 % NaCl (w/v, optimum 2 %). Analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Saccharospirillum, as the highest sequence similarities were observed with Saccharospirillum impatiens EL-105T (96.46 %), Saccharospirillum salsuginis YIM-Y25T (96.32 %) and Saccharospirillum aestuariiIMCC 4453T (95.17 %). The next closest matches were with other genera and below 95.0 %. Phylogenetic analyses revealed that the strain forms a robust clade with other species of the genus Saccharospirillum. The main respiratory quinone was Q-8 and the major fatty acids were C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The DNA G+C content was 55.2 mol%. Molecular, physiological and biochemical differences between strain CPA1T and other type strains of species of the genus Saccharospirillum support the addition of this novel species to the genus, and the name Saccharospirillum correiae sp. nov. is proposed, with CPA1T (=CECT 9131T=LMG 29516T) as the type strain.

Altererythrobacter halimionae sp. nov. and Altererythrobacter endophyticus sp. nov., two endophytes from the salt marsh plant Halimione portulacoides.

Two Gram-negative, rod-shaped, motile bacterial strains, named CPA5T and BR75T, were isolated from the halophyte Halimione portulacoides. Both presented optimum growth at 30 °C, pH 7.0-7.5 and 1-2 % NaCl (w/v) for strain CPA5T, and pH 7.5-8.0 and 2 % NaCl (w/v) for strain BR75T. Phylogenetic analyses based on 16S rRNA gene sequences affiliated both strains to the genus Altererythrobacter. CPA5T presented highest 16S rRNA gene sequence similarity with Altererythrobacter aestuarii KYW147T (96.5 %), followed by Altererythrobacter namhicola KYW48T (95.9 %), Novosphingobium indicum H25T (95.6 %) and Altererythrobacter oceanensis Y2T (95.5 %). BR75T displayed highest similarity with Altererythrobacter marensis MSW-14T (96.5 %), followed by Altererythrobacter xinjiangensis S3-63T, Altererythrobacter luteolus SW-109T and Altererythrobacter indicus MSSRF26T (96.1 %). Neither strain contained Bacteriochlorophyll a. The main fatty acids observed for CPA5T were C17 : 1ω6c and summed features 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and 8 (C18 : 1 ω7c and/or C18 : 1ω6c). The latter summed feature was the dominant fatty acid observed for strain BR75T as well. The major polar lipids were phosphatidylethanolamine, unidentified phospholipids and unidentified glycolipids for both strains. The predominant ubiquinone was Q-10 for both strains, and the DNA G+C contents were 63.4 mol% and 58.3 mol% for CPA5T and BR75T, respectively. Based on phenotypic and genotypic results, both strains represent novel species belonging to the genus Altererythrobacter for which the names Altererythrobacter halimionae sp. nov. (type strain CPA5T=CECT 9130T=LMG 29519T) and Altererythrobacter endophyticus sp. nov (type strain BR75T=CECT 9129T=LMG 29518T) are proposed.

Culture-independent methods reveal high diversity of OXA-48-like genes in water environments.

The carbapenemase OXA-48 was identified for the first time in 2001 and is now one of the greatest concerns in terms of antibiotic resistance. While many studies report clinical OXA-48-like producers, few reports refer blaOXA-48-like genes in environmental bacteria. The main goal of this study was to evaluate the diversity of blaOXA-48-like genes in aquatic systems, using culture-independent approaches. For that, environmental DNA was obtained from riverine and estuarine water and used to construct clone libraries of blaOXA-48-like gene polymerase chain reaction amplicons. blaOXA-48-like libraries from river and estuarine water DNA comprised 75 and 70 clones, respectively. Sequence analysis showed that environmental blaOXA-48-like genes show a broader diversity than that so far observed in clinical settings. In total, 50 new OXA-48 variants were identified as well as sequences identical to previously reported OXA-48, OXA-181, OXA-199, OXA-204 and OXA-162. Though we have no evidence that these genes were carried by bacteria that are members of the natural heterotrophic flora or bacteria that have entered this particular water environment through anthropogenic sources, these results reinforce the role of aquatic systems as antibiotic resistance reservoirs. The variants of blaOXA-48 here described should be taken into account when designing molecular strategies for detecting this gene.

Microbacterium diaminobutyricum sp. nov., isolated from Halimione portulacoides, which contains diaminobutyric acid in its cell wall, and emended description of the genus Microbacterium.

Three actinobacterial strains were isolated from roots of the salt-marsh plant Halimione portulacoides collected in Ria de Aveiro, Portugal. Molecular typing using enterobacterial repetitive intergenic consensus ERIC-PCR fingerprinting showed the strains to be highly similar. Phylogenetic analyses based on the 16S rRNA gene sequence and multilocus sequence analysis (MLSA) using gyrB, rpoB, recA and ppk and 16S rRNA genes sequences showed that the strains represented a member of the genus Microbacterium, with Microbacterium lacus DSM 18910T as the closest phylogenetic relative. DNA-DNA hybridization between strain RZ63T and its closest relative was below 70 %, supporting the hypothesis that it represented a distinct genomic species. Chemotaxonomic analyses of the novel strains and their DNA G+C contents confirmed their affiliation to the genus Microbacterium, however, the peptidoglycan of RZ63T contained diaminobutyric acid as the diagnostic diamino acid. In addition, physiological and fatty acid analyses revealed differences between these strains and their phylogenetic relatives, reinforcing their status as a distinct species. Based on the physiological, genetic and chemotaxonomic characterisation it is proposed that the strains studied represent a novel species of the genus Microbacterium for which the name Microbacterium diaminobutyricum sp. nov. is proposed (type strain RZ63T=DSM 27101T=CECT 8355T).

Intralesional Patterns of MRI ADC Maps Predict Outcome in Experimental Stroke.

BACKGROUND: After acute ischemia, the tissue that is at risk of infarction can be detected by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch but the time that is needed to process PWI limits its use. As DWI is highly sensitive to acute ischemic tissue damage, we hypothesized that different ADC patterns represent areas with a different potential for recovery. METHODS: In a model of permanent middle cerebral artery occlusion (pMCAO), Sprague-Dawley rats were randomly distributed to sham surgery and pMCAO. We further separated the pMCAO group according to intralesional ADC pattern (homogeneous or heterogeneous). At 24 h after ischemia induction, we analyzed lesion size, functional outcome, cell death expression, and brain protection markers including ROS enzyme NOX-4. MRI included DWI (ADC maps), DTI (tractography), and PWI (CBF, CBV and MTT). RESULTS: The lesion size was similar in pMCAO rats. Animals with a heterogeneous pattern in ADC maps showed better functional outcome in Rotarod test (p = 0.032), less expression of cell death (p = 0.014) and NOX-4 (p = 0.0063), higher intralesional CBF (p = 0.0026) and larger PWI/DWI mismatch (p = 0.007). CONCLUSIONS: In a rodent model for ischemic stroke, intralesional heterogeneity in ADC maps was related to better functional outcome in lesions of similar size and interval after pMCAO. DWI ADC maps may assist in the early identification of ischemic tissue with an increased potential for recovery as higher expression of acute protection markers, lower expression of cell death, increased PWI/DWI mismatch, and higher intralesional CBF were present in animals with a heterogeneous ADC pattern.

Combined effect of temperature and copper pollution on soil bacterial community: climate change and regional variation aspects.

The aim of this study was to assess the combined effects of temperature and copper (Cu) contamination in the structure of soil bacterial community. For this, contaminated or spiked and control soils from two different geographic origins (PT-Portugal and DK-Denmark) were used. The DK soil was from a historically contaminated study field, representing a long-term exposure to Cu while the PT soil was from a clean site and freshly spiked with Cu. Soil bacterial communities were exposed in mesocosms during 84 days to 3 different temperatures based on values typically found in each geographic region and temperature conditions that simulated a warming scenario. Obtained results indicate that Cu stress alters the structure of bacterial community and that this effect is, to some extent, temperature-dependent. Effects on bacterial diversity for both soils were also observed. Differences in the DK and PT communities' response were apparent, with the community from the historically contaminated soil being more resilient to temperature fluctuations. This study presents evidence to support the hypothesis that temperature alters the effect of metals on soils. Further, our results suggest that the definition of soils quality criteria must be based on studies performed under temperatures selected for the specific geographic region. Studies taking into account temperature changes are needed to model and predict risks, this is important to e.g. future adjustments of the maximum permissible levels for soil metal contamination.

Assessment of rhizospheric culturable bacteria of Phragmites australis and Juncus effusus from polluted sites.

This study aimed at the isolation and characterization of metal(loid)-tolerant bacteria from the rhizosphere of Phragmites australis and Juncus effusus plants growing in two long-term contaminated sites in Northern Portugal. Site 1 had higher contamination than Site 3. Bacteria were isolated using metal(loid)-supplemented (Cd, Zn, and As) media. Isolates were grouped by random amplified polymorphic DNA and identified by 16S rRNA gene sequencing. Strains were also examined for their metal(loid) tolerance. The counts of metal(loid)-tolerant bacteria were higher in Site 1 and ranged between log 7.17 CFU g(-1) soil in As-containing medium and log 7.57 CFU g(-1) soil in Zn-containing medium, while counts at Site 3 varied between log 5.33 CFU g(-1) soil in Cd-containing medium and log 6.97 CFU g(-1) soil in As-containing medium. The composition of bacterial populations varied between locations. In Site 1, the classes Actinobacteria (36%) and Bacilli (24%) were well represented, while in Site 3 strains were mainly affiliated to classes Actinobacteria (35%), γ-Proteobacteria (35%), and ß-Proteobacteria (12%). The order of metal(loid) toxicity for the isolated strains was Cd > As > Zn. Overall, 10 strains grew at 500 mg Cd L(-1) , 1000 mg Zn L(-1) , and 500 mg As L(-1) , being considered the most metal(loid)-tolerant bacteria. These strains belonged to genera Cupriavidus, Burkholderia, Novosphingobium, Sphingobacterium, Castellaniella, Mesorhizobium, Chryseobacterium, and Rhodococcus and were mainly retrieved from Site 1. The multiple metal(loid)-tolerant strains isolated in this study have potential to be used in bioremediation/phytoremediation.

A microcosm approach to evaluate the degradation of tributyltin (TBT) by Aeromonas molluscorum Av27 in estuarine sediments.

Tributyltin (TBT) is a biocide extremely toxic to a wide range of organisms, which has been used for decades in antifouling paints. Despite its global ban in 2008, TBT is still a problem of great concern due to the high levels trapped in sediments. Aeromonas molluscorum Av27 is a TBT degrading bacterium that was isolated from an estuarine system. We investigated the ability and the role of this bacterium on TBT degradation in this estuarine system, using a microcosm approach in order to mimic environmental conditions. The experiment was established and followed for 150 days. Simultaneously, changes in the indigenous bacterial community structure were also investigated. The results revealed a maximum TBT degradation rate of 28% accompanied by the detection of the degradation products over time. Additionally, it was observed that TBT degradation was significantly enhanced by the presence of Av27. In addition a significantly higher TBT degradation occurred when the concentration of Av27 was higher. TBT degradation affected the bacterial community composition as revealed by the changes in the prevalence of Proteobacteria subdivisions, namely the increase of Deltaproteobacteria and the onset of Epsilonproteobacteria. However, the addition of Av27 strain did not affect the dominant phylotypes. Total bacterial number, bacterial biomass productivity, 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analyses also indicated alterations on the bacterial community structure over time, with bacteria non-tolerant to pollutants increasing their representativeness, as, for instance, the increase of the number of Alphaproteobacteria clones from 6% in the beginning to 12% at the end of the experiment. The work herein presented confirms the potential of Av27 strain to be used in the decontamination of TBT-polluted environments.