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.

Hospital microbial surface colonization revealed during monitoring of Klebsiella spp., Pseudomonas aeruginosa, and non-tuberculous mycobacteria.

Hospital environmental conditions, human occupancy, and the characteristics of the equipment influence the survival of microbial communities and raise a concern with regard to nosocomial infections. The objective of the present work was to use the monitoring of Pseudomonas aeruginosa, Klebsiella spp. and non-tuberculous mycobacteria as a strategy to improve knowledge on microbial colonization of non-critical equipment and surfaces, in a tertiary hospital from Central Portugal. A 3-month microbiological survey was performed in a district teaching hospital. A total of 173 samples were obtained from the wards Hematology, Urology, Medicine, and Renal Transplants, and 102 presumptive strains recovered. Per sampling, Pseudomonas Isolation agar showed 42.8 to 73.3% of presumptive P. aeruginosa colonies and MacConkey agar recovered mostly Staphylococcus. Most of the colonies recovered in Middlebrook 7H10-PANTA belonged to the genus Methylobacterium. Taps and WC shower curtains carry high bacterial species diversity. The Redundancy Analysis grouped the samples in those mostly handled by patients, and those mostly handled by healthcare staff or of mixed use. This study shows that the preferential users of the space and equipment seem to be important contributors to the microbial community. The most recovered genus was Methylobacterium, known as colonizer of the water distribution system therefore, it is possible that the water points and biofilms in taps also contribute as dispersion hotspots.

Persistence of microbial communities including Pseudomonas aeruginosa in a hospital environment: a potential health hazard.

BACKGROUND: The persistence of microbial communities and how they change in indoor environments is of immense interest to public health. Moreover, hospital acquired infections are significant contributors to morbidity and mortality. Evidence suggests that, in hospital environments agent transfer between surfaces causes healthcare associated infections in humans, and that surfaces are an important transmission route and may act as a reservoir for some of the pathogens.This study aimed to evaluate the diversity of microorganisms that persist on noncritical equipment and surfaces in a main hospital in Portugal, and are able to grow in selective media for Pseudomonas, and relate them with the presence of Pseudomonas aeruginosa. RESULTS: During 2 years, a total of 290 environmental samples were analyzed, in 3 different wards. The percentage of equipment in each ward that showed low contamination level varied between 22% and 38%, and more than 50% of the equipment sampled was highly contaminated. P. aeruginosa was repeatedly isolated from sinks (10 times), from the taps' biofilm (16 times), and from the showers and bedside tables (two times). Two ERIC clones were isolated more than once. The contamination level of the different taps analyzed showed correlation with the contamination level of the hand gels support, soaps and sinks. Ten different bacteria genera were frequently isolated in the selective media for Pseudomonas. Organisms usually associated with nosocomial infections as Stenotrophomonas maltophilia, Enterococcus feacalis, Serratia nematodiphila were also repeatedly isolated on the same equipment. CONCLUSIONS: The environment may act as a reservoir for at least some of the pathogens implicated in nosocomial infections. The bacterial contamination level was related to the presence of humidity on the surfaces, and tap water (biofilm) was a point of dispersion of bacterial species, including potentially pathogenic organisms. The materials of the equipment sampled could not be related to the microbial contamination level. The presence of a disinfectant in the isolation medium suggests that the number of microorganism in the environment could be higher and shows the diversity of disinfectant resistant species. The statistical analysis suggests that the presence of bacteria could increase the risk of transmission by hand manipulation.

Post-measurement compressed calibration for ICP-MS-based metal quantification in mine residues bioleaching.

Bioleaching is an actual economical alternative to treat residues, which allows, depending on the chosen strategy, two possible outcomes: (1) a leachate enriched with target metals, or (2) a residue enriched in target metals through the leaching of interfering components (IC). This work aimed to study the metals released by bioprocessing the Panasqueira mine tailings, as a strategy to increase critical metals' relative concentration in residues. Biostimulation of the local microbiota was compared to a bioaugmentation approach using the autochthonous Diaphorobacter polyhydroxybutyrativorans strain B2A2W2. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was selected to study the metals released in the leachate through multi-element external standards. A new data treatment method was developed to use a preliminary sweep of intensities to quantify the non-initial target metals concentration in the leachate, based on preliminary ICP-MS intensity measurements. The results demonstrated that biostimulation was an efficient bioleaching strategy for the IC silicon, aluminium, magnesium, selenium, manganese, zinc, iron, and copper, by decreasing concentration, resulting in a relative increase in the gallium and yttrium (10x) levels in the treated residue. The strategy followed to quantify a large number of elements with ICP-MS using a reduced number of data points for calibration proved valid and speeded up the analytical process.

Chromium resistance strategies and toxicity: what makes Ochrobactrum tritici 5bvl1 a strain highly resistant.

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.

Isolation and characterization of bacteria resistant to metallic copper surfaces.

Metallic copper alloys have recently attracted attention as a new antimicrobial weapon for areas where surface hygiene is paramount. Currently it is not understood on a molecular level how metallic copper kills microbes, but previous studies have demonstrated that a wide variety of bacteria, including Escherichia coli, Staphylococcus aureus, and Clostridium difficile, are inactivated within minutes or a few hours of exposure. In this study, we show that bacteria isolated from copper alloy coins comprise strains that are especially resistant against the toxic properties exerted by dry metallic copper surfaces. The most resistant of 294 isolates were Gram-positive staphylococci and micrococci, Kocuria palustris, and Brachybacterium conglomeratum but also included the proteobacterial species Sphingomonas panni and Pseudomonas oleovorans. Cells of some of these bacterial strains survived on copper surfaces for 48 h or more. Remarkably, when these dry-surface-resistant strains were exposed to moist copper surfaces, resistance levels were close to those of control strains and MICs for copper ions were at or below control strain levels. This suggests that mechanisms conferring resistance against dry metallic copper surfaces in these newly isolated bacterial strains are different from well-characterized copper ion detoxification systems. Furthermore, staphylococci on coins did not exhibit increased levels of resistance to antibiotics, arguing against coselection with copper surface resistance traits.

Different physiological responses to chromate and dichromate in the chromium resistant and reducing strain Ochrobactrum tritici 5bvl1.

Studies of Cr(VI) toxicity are generally performed using chromate salts in solution, both when studying the effects on prokaryotes and eukaryotes. Some studies on human carcinogenesis and toxicology on bacteria were done using dichromate, but comparison with chromate was never reported before, and dichromate existence was never taken into consideration and usually overlooked. This paper studied comparatively the effect of dichromate and chromate on the physiology of Ochrobactrum tritici strain 5bvl1, a highly Cr(VI)-resistant and reducing microorganism. This study demonstrated that the addition of chromate or dichromate sodium salts to growth medium at neutral pH ended-up in two different solutions with a different balance of chemical species. Cr(VI) was toxic to O. tritici strain 5bvl1, as clearly shown on growth, reduction, respiration, glucose accumulation assays and by comparing cell morphology. Moreover, the addition of sodium dichromate was always more toxic to cells when compared to chromate and achieved a higher inhibition of every parameter studied. The toxicity differences between the two Cr(VI) oxyanions indicate the possibility of a different impact of Cr(VI) contamination on the environment. This may be of major importance, considering the slight acidity of most of the arable lands which favours the presence of dichromate, the more toxic species.

Identification of an aox system that requires cytochrome c in the highly arsenic-resistant bacterium Ochrobactrum tritici SCII24.

Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici DeltaaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.

Metal-induced phosphate extracellular nanoparticulate formation in Ochrobactrum tritici 5bvl1.

Hexavalent chromium (Cr(VI)) is a toxic environmental contaminant which detoxification consists in reduction to Cr(III). In this work, the Cr(VI)-resistant and reducing Ochrobactrum tritici 5bvl1 produced phosphate nanoparticles upon exposure to Cr(VI) and Fe(III), effectively removing chromium from solution. Under Cr(VI) stress, higher siderophore production by strain 5bvl1 was observed. Cr(VI) toxicity was decreased in presence of Fe(III), increasing the growth and Cr(VI)-reduction rates in cell cultures, lowering the amount of morphologically compromised cells and promoting chromium immobilization as insoluble extracellular phosphate complexes. The formation of phosphate nanoparticles increased with Cr(VI) and Fe(III) concentrations and was also stimulated by Ni(II). Under these experimental conditions, nanoparticle formation occurred together with enhanced inorganic phosphate consumption by cells and increased polyphosphate kinase (PPK) activity. NMR analysis of the particles showed the presence of both polyphosphate and phosphonate together with orthophosphate, and FT-IR supported these results, also showing evidences of Cr(III) coordination. This work demonstrated that O. tritici 5bvl1 possesses protection mechanisms against chromium toxicity other than the presence of the Cr(VI) pump and SOD related enzymes previously described. Future assessment of the molecular regulation of production of these nanoparticles will open new perspectives for remediation of metal contaminated environments.