Isolation and Characterization of Two Novel Bacteria Afipia cberi and Mesorhizobium hominis from Blood of a Patient Afflicted with Fatal Pulmonary Illness

We recently isolated and discovered new Bradyrhizobiaceae microbes from the cryopreserved culture broth of blood samples from 3 patients with poorly defined illnesses using modified SP4 media and culture conditions coupled with genomic sequencing. Using a similar protocol, we studied a previously cryopreserved culture broth of blood sample from a patient who had succumbed to an acute onset of fulminant pulmonary illness. We report that two phases of microbial growth were observed in the re-initiated culture. Biochemical and genomic characterization revealed microbes isolated from the first phase of growth were new Afipia species of Bradyrhizobiaceae, tentatively named A. cberi with a ~ 5 MB chromosome that was different from those of all previously known Afipia microbes including the newly discovered A. septicemium. The microbes isolated from the second phase of growth were prominent sugar assimilators, novel Phyllobacteriaceae, phylogenetically most closely related to Mesorhizobium and tentatively named M. hominis with a ~ 5.5 MB chromosome. All A. cberi isolates carry a circular ~ 140 KB plasmid. Some M. hominis isolates possess a circular ~ 412 KB plasmid that can be lost in prolonged culture or passage. No antibiotics resistant genes could be identified in both of the A. cberi and M. hominis plasmids. Antibiotic susceptibility studies using broth culture systems revealed isolates of A. cberi could be sensitive to some antibiotics, but all isolates of M. hominis were resistant to essentially all tested antibiotics. However, the cell-free antibiotics susceptibility test results may not be applicable to clinical treatment against the microbes that are known to be capable of intracellular growth. It remains to be determined if the 2 previously unknown Rhizobiales were indeed pathogenic and played a role in the pulmonary disease process in this patient. Specific probes and methods will be developed to re-examine the diseased lungs from patient''s autopsy.     

Variability in Effectiveness of Rhizobia during Culture and in Nodules

The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.     

Comparison of Two Liquid Blood Culture Media Containing Sodium Polyanetholesulfonate: Tryptic Soy and Columbia

In a comparison of tryptic soy broth and Columbia broth, two blood culture media containing sodium polyanetholesulfonate, there were 589 positive cultures (excluding presumed contaminants). The two media were equivalent in performance except for lower detection rates for Staphylococcus aureus (P < 0.01) and Pseudomonas aeruginosa (P = 0.05) and a higher detection rate for Bacillus (P < 0.01) in Columbia broth. No significant differences were noted in time intervals to detection of positivity. Routine subcultures on the 1st and 5th days of incubation provided the initial detection of 18.1% of the positive cultures.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Growth Characteristics, Bile Sensitivity, and Freeze Damage in Colonial Variants of Lactobacillus acidophilus

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneous culture of Lactobacillus acidophilus RL8K. R and S types were stable upon repeated transfer on agar, but revertant colonies did appear after broth transfers. When propagated in commercial MRS broth, R and S cultures showed similar growth characteristics, and both cell types were insensitive to freezing and frozen storage at −20°C. Alternatively, during growth in scratch MRS broth, R cultures shifted to a reduced rate of growth during the late logarithmic phase. R cells grown under these conditions were susceptible to death by freezing and injury at −20°C. Microscopically, R cells were observed as long gram-positive rods with small nonstainable blebs protruding from the cell wall. In bile sensitivity studies of R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1% bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bile resistance and freeze damage of R and S cells suggest that colonial and cellular morphologies are important considerations for the selection of Lactobacillus strains as dietary adjuncts and for the development of growth conditions for preparing frozen concentrated cultures from either cell type.     

A Novel Strain D5 Isolated from Acacia confusa

We isolated a novel strain D5 from nodules of Acacia confusa. Under strict sterile conditions the strain could successfully nodulate Acacia confusa, A. crassicarpa and A. mangium, with nitrogenase activity ranging from 18.90 to 19.86 nmol·g⁻¹·min⁻¹. In the phylogenetic tree based on a complete 16S rRNA gene sequence, the sequence of strain D5 shared 99% homology with that of four species of genus Pseudomonas. The 685 bp nodA fragment amplified from strain D5 shared 95% homology with the nodA sequence of 9 species of genus Bradyrhizobium, with a genetic distance of 0.01682. The 740 bp nifH gene fragment was amplified from strain D5. This strain D5 nifH gene and Bradyrhizobium spp. formed a branch, showing 98% homology and a genetic distance of 0. The homology between this branch and the Bradyrhizobium spp. DG in another branch was 99%, with a genetic distance of 0.007906. These results indicate that this strain D5 is a new type of nitrogen-fixing bacterium.     

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.     

Calcium-Induced Alteration of Cellular Morphology Affecting the Resistance of Lactobacillus acidophilus to Freezing

Examination of factors affecting the resistance of Lactobacillus acidophilus NCFM culture concentrates to freeze injury induced during frozen storage at -20°C revealed that calcium supplementation of the growth medium contributed to the storage stability of cells prepared in static culture. Culture concentrates of L. acidophilus NCFM were prepared from cells propagated in MRS broth or MRS broth supplemented with 0.1% calcium carbonate, calcium chloride, or calcium phosphate. After 28 days of frozen storage at -20°C, concentrated cells (3.2 × 10⁹ colony-forming units per ml) prepared from MRS broth cultures showed an 84% reduction in viable cells. Of the remaining viable cells, 88% were sublethally injured and unable to form colonies on MRS agar supplemented with 0.15% bile. Cells prepared in calcium-supplemented MRS broths demonstrated more resistance to frozen storage. Viability and injury losses in the frozen concentrates were limited to 10 to 39% and 3 to 23%, respectively. It was observed that calcium supplementation of MRS medium resulted in a morphological transition of L. acidophilus NCFM from filamentous to bacilloid rods, and the bacilloid cells were more resistant to freezing and storage at conventional freezer temperatures. The results suggest that the morphology of the L. acidophilus cell may be an important consideration in the preparation of freeze-stable culture concentrates.     

Common Contaminants in Next-Generation Sequencing That Hinder Discovery of Low-Abundance Microbes

Unbiased high-throughput sequencing of whole metagenome shotgun DNA libraries is a promising new approach to identifying microbes in clinical specimens, which, unlike other techniques, is not limited to known sequences. Unlike most sequencing applications, it is highly sensitive to laboratory contaminants as these will appear to originate from the clinical specimens. To assess the extent and diversity of sequence contaminants, we aligned 57 “1000 Genomes Project” sequencing runs from six centers against the four largest NCBI BLAST databases, detecting reads of diverse contaminant species in all runs and identifying the most common of these contaminant genera (Bradyrhizobium) in assembled genomes from the NCBI Genome database. Many of these microorganisms have been reported as contaminants of ultrapure water systems. Studies aiming to identify novel microbes in clinical specimens will greatly benefit from not only preventive measures such as extensive UV irradiation of water and cross-validation using independent techniques, but also a concerted effort to sequence the complete genomes of common contaminants so that they may be subtracted computationally.     

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched in Bolton broth and Preston broth, followed by plating on Skirrow, mCCDA, and blood agar (a membrane filter on its surface) media, respectively. Thirty-nine of 99 samples were positive and 71 Campylobacter were isolated by one or more methods. More than one species of Campylobacter were obtained in 8 (20.5 %) of 39 positive samples and two genotypes were yielded on the same medium (11 samples, 28.2 %) by pulsed-field gel electrophoresis (PFGE) genotyping. Enrichment by Preston broth was significantly better than by Bolton broth (P?<?0.05). Moreover, the latter failed to detect Campylobacter jejuni strains. Skirrow medium was significantly less efficient than mCCDA medium and membrane filtration method (P?<?0.05). Overall, the combination of PC (primary enrichment in Preston broth, followed by selective enrichment on mCCDA agar), PF (primary enrichment in Preston broth, followed by membrane filtration culture onto blood agar), and BF (primary enrichment in Bolton broth, followed by membrane filtration culture onto blood agar) methods provided the optimum isolation rate of Campylobacter spp.     

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires'' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm⁺, showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm⁺ background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.     

Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.     

Evaluating the Use of Blood Cultures in the Management of Children Hospitalized for Community-Acquired Pneumonia

BackgroundBlood cultures are often recommended for the evaluation of community-acquired pneumonia (CAP). However, institutions vary in their use of blood cultures, and blood cultures have unclear utility in CAP management in hospitalized children.ObjectiveTo identify clinical factors associated with obtaining blood cultures in children hospitalized with CAP, and to estimate the association between blood culture obtainment and hospital length of stay (LOS).MethodsWe performed a multicenter retrospective cohort study of children admitted with a diagnosis of CAP to any of four pediatric hospitals in the United States from January 1, 2011-December 31, 2012. Demographics, medical history, diagnostic testing, and clinical outcomes were abstracted via manual chart review. Multivariable logistic regression evaluated patient and clinical factors for associations with obtaining blood cultures. Propensity score-matched Kaplan-Meier analysis compared patients with and without blood cultures for hospital LOS.ResultsSix hundred fourteen charts met inclusion criteria; 390 children had blood cultures obtained. Of children with blood cultures, six (1.5%) were positive for a pathogen and nine (2.3%) grew a contaminant. Factors associated with blood culture obtainment included presenting with symptoms of systemic inflammatory response syndrome (OR 1.78, 95% CI 1.10–2.89), receiving intravenous hydration (OR 3.94, 95% CI 3.22–4.83), receiving antibiotics before admission (OR 1.49, 95% CI 1.17–1.89), hospital admission from the ED (OR 1.65, 95% CI 1.05–2.60), and having health insurance (OR 0.42, 95% CI 0.30–0.60). In propensity score-matched analysis, patients with blood cultures had median 0.8 days longer LOS (2.0 vs 1.2 days, P < .0001) without increased odds of readmission (OR 0.94, 95% CI 0.45–1.97) or death (P = .25).ConclusionsObtaining blood cultures in children hospitalized with CAP rarely identifies a causative pathogen and is associated with increased LOS. Our results highlight the need to refine the role of obtaining blood cultures in children hospitalized with CAP.     

Growth, Respiration, and Polypeptide Patterns of Bradyrhizobium sp. (Arachis) Strain 3G4b20 from Succinate- or Oxygen-Limited Continuous Cultures

Succinate- or oxygen-limited continuous cultures were used to study the influences of different concentrations of dissolved oxygen and ammonia on the growth, respiration, and polypeptide patterns of Bradyrhizobium sp. (Arachis) strain 3G4b20. During succinate-limited growth, molar growth yields on succinate (Y_succ) ranged from 38.9 to 44.4 g (dry weight) of cells mol of succinate⁻¹ and were not greatly influenced by changes in dilution rates or changes in the oxygen concentrations that we tested. Succinate, malate, and fumarate induced the highest rates of oxygen uptake in all of the steady states in which the supply rates of (NH₄)₂SO₄ ranged between 322 and 976 μmol h⁻¹. However, the amino acids aspartate, asparagine, and glutamate could also be used as respiratory substrates, especially when the (NH₄)₂SO₄ supply rate was decreased to 29 μmol h⁻¹. Glutamine-dependent respiration was seen only when the (NH₄)₂SO₄ supply rate was 29 μmol h⁻¹ and thus appears to be under tight ammonia control. Nitrogenase activity was detected only when the culture was switched from a succinate-limited steady state to an oxygen-limited steady state. Comparison of major silver-stained proteins from three steady states by two-dimensional gel electrophoresis revealed that nearly 60% were affected by oxygen and 24% were affected by ammonia. These data are consistent with reports that oxygen has a major regulatory role over developmental processes in Rhizobium sp. and Bradyrhizobium sp.     

Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.     

Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

Background

Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1–3 days subculture step currently required before any therapeutic adjustments can be made.

Methodology/Principal Findings

Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000–7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained.

Conclusions/Significance

Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia.     

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.