Real-Life Experience and Diagnostic Utility of the BioFire Joint Infection PCR Panel in Bone and Joint Infections: Analysis of a Prospective Validation Study.

INTRODUCTION: Microbiological diagnosis is central for adequate treatment of bone and joint infections. Culture-based methods have a limited diagnostic sensitivity and a long turnaround time (TAT). The objective of this study was to compare the diagnostic performance of BioFire Joint Infection Panel Investigational Use Only version (hereafter BioFire)-a sample-to-result multiplex PCR panel-with culture-based methods and 16S ribosomal RNA (rRNA) PCR and sequencing, when available. METHODS: This study presents a retrospective analysis of a prospective validation study of the BioFire panel. Specimens were obtained from consecutive patients evaluated for suspected bone and joint infections and processed using culture, BioFire, and 16S rRNA PCR and sequencing. Final clinical diagnosis was used as the reference for definition of infection. RESULTS: Samples, including synovial fluid, bone and periarticular tissue, were obtained from 57 patients, 39 of whom were finally diagnosed with a bone or joint infection. Cultures were positive in 27/39 infected patients and in 3/18 uninfected patients (sensitivity 69%, specificity 83%). BioFire was positive in 22/39 infected patients and in none of the uninfected patients (sensitivity 56%, specificity 100%). Sensitivity for PCR panel organisms was 92% (22/24) and sensitivity for organisms identified by any microbiological modality was 69% (22/32). Gram stain results were positive in 13/39 infected patients and in none of the uninfected patients (sensitivity 33%, specificity 100%). 16S rRNA was positive in 20/28 infected patients and in 0/12 uninfected patients (sensitivity 71%, specificity 100%). Net machine time for BioFire-1 h-was shorter than the mean TAT for Gram stain results, which was 4 h. CONCLUSION: BioFire offered equivalent diagnostic performance with superior TAT for bone and joint infections, compared with conventional methods.

Diversity and Differentiation of Duckweed Species from Israel.

Duckweeds (Lemnaceae) are tiny plants that float on aquatic surfaces and are typically isolated from temperate and equatorial regions. Yet, duckweed diversity in Mediterranean and arid regions has been seldom explored. To address this gap in knowledge, we surveyed duckweed diversity in Israel, an ecological junction between Mediterranean and arid climates. We searched for duckweeds in the north and center of Israel on the surface of streams, ponds and waterholes. We collected and isolated 27 duckweeds and characterized their morphology, molecular barcodes (atpF-atpH and psbK-psbI) and biochemical features (protein content and fatty acids composition). Six species were identified-Lemna minor, L. gibba and Wolffia arrhiza dominated the duckweed populations, and together with past sightings, are suggested to be native to Israel. The fatty acid profiles and protein content further suggest that diverged functions have attributed to different haplotypes among the identified species. Spirodela polyrhiza, W. globosa and L. minuta were also identified but were rarer. S. polyrhiza was previously reported in our region, thus, its current low abundance should be revisited. However, L. minuta and W. globosa are native to America and Far East Asia, respectively, and are invasive in Europe. We hypothesize that they may be invasive species to our region as well, carried by migratory birds that disperse them through their migration routes. This study indicates that the duckweed population in Israel's aquatic environments consists of both native and transient species.

Comparative Analysis of Volatiles Emitted from Tomato and Pepper Plants in Response to Infection by Two Whitefly-Transmitted Persistent Viruses.

The whitefly Bemisia tabaci is one of the most important agricultural pests due to its extreme invasiveness, insecticide resistance, and ability to transmit hundreds of plant viruses. Among these, Begomoviruses and recombinant whitefly-borne Poleroviruses are transmitted persistently. Several studies have shown that upon infection, plant viruses manipulate plant-emitted volatile organic compounds (VOCs), which have important roles in communication with insects. In this study, we profiled and compared the VOCs emitted by tomato and pepper plant leaves after infection with the Tomato yellow leaf curl virus (TYLCV) (Bogomoviruses) and the newly discovered Pepper whitefly-borne vein yellows virus (PeWBVYV) (Poleroviruses), respectively. The results identified shared emitted VOCs but also uncovered unique VOC signatures for each virus and for whitefly infestation (i.e., without virus infection) independently. The results suggest that plants have general defense responses; however, they are also able to respond individually to infection with specific viruses or infestation with an insect pest. The results are important to enhance our understanding of virus- and insect vector-induced alteration in the emission of plant VOCs. These volatiles can eventually be used for the management of virus diseases/insect vectors by either monitoring or disrupting insect-plant interactions.

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation.

The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus's recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo.

The 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

Investigation of Outbreaks of Extended-Spectrum Beta-Lactamase-Producing Klebsiella Pneumoniae in Three Neonatal Intensive Care Units Using Whole Genome Sequencing.

Infections caused by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are on a constant rise and are a noted cause of outbreaks in neonatal intensive care units (NICUs). We used whole genome sequencing (WGS) to investigate the epidemiology of consecutive and overlapping outbreaks caused by ESBL-KP in NICUs in three hospitals in close proximity. Clonality of 43 ESBL-KP isolates from 40 patients was determined by BOX-PCR. Short-read sequencing was performed on representative isolates from each clone. The dominant clones from each NICU were sequenced using long-read sequencing. Bioinformatics methods were used to define multilocus sequence type (MLST), analyze plasmid content, resistomes, and virulence factors. In each NICU, we found a unique dominant clone (ST985, ST37, and ST35), each belonging to a distinct sequence type (ST), as well as satellite clones. A satellite strain in NICU-2 (ST35) was the dominant strain in NICU-3, where it was isolated four weeks later, suggesting transmission. NICU-1- and NICU-2-dominant strains had blaCTX-M-15 carried on a similar transposable element (Tn3-ISEcp1) but at different locations: on a plasmid and on the chromosome, respectively. We concluded that the overlapping ESBL-KP outbreaks were a combination of clonal transmission within NICUs, possible transposable element transmission between NICUs, and repeated importation of ESBL-KP from the community.

High Resolution Proteome of Lipid Droplets Isolated from the Pennate Diatom Phaeodactylum tricornutum (Bacillariophyceae) Strain pt4 provides mechanistic insights into complex intracellular coordination during nitrogen deprivation.

Lipid droplets (LDs) are an organelle conserved amongst all eukaryotes, consisting of a neutral lipid core surrounded by a polar lipid monolayer. Many species of microalgae accumulate LDs in response to stress conditions, such as nitrogen starvation. Here, we report the isolation and proteomic profiling of LD proteins from the model oleaginous pennate diatom Phaeodactylum tricornutum, strain Pt4 (UTEX 646). We also provide a quantitative description of LD morphological ontogeny, and fatty acid content. Novel cell disruption and LD isolation methods, combined with suspension-trapping and nanoflow liquid chromatography coupled to high resolution mass spectrometry, yielded an unprecedented number of LD proteins. Predictive annotation of the LD proteome suggests a broad assemblage of proteins with diverse functions, including lipid metabolism and vesicle trafficking, as well as ribosomal and proteasomal machinery. These proteins provide mechanistic insights into LD processes, and evidence for interactions between LDs and other organelles. We identify for the first time several key steps in diatom LD-associated triacylglycerol biosynthesis. Bioinformatic analyses of the LD proteome suggests multiple protein targeting mechanisms, including amphipathic helices, post-translational modifications, and translocation machinery. This work corroborates recent findings from other strains of P. tricornutum, other diatoms, and other eukaryotic organisms, suggesting that the fundamental proteins orchestrating LDs are conserved, and represent an ancient component of the eukaryotic endomembrane system. We postulate a comprehensive model of nitrogen starvation-induced diatom LDs on a molecular scale, and provide a wealth of candidates for metabolic engineering, with the potential to eventually customize LD contents.

Screening asymptomatic households for Streptococcus pyogenes pharyngeal carriage as a part of in-hospital investigation of puerperal sepsis.

BACKGROUND: Invasive group A streptococcal (iGAS) infection in the peripartum setting is a rare but devastating disease occasionally occurring as a health care-associated infection (HAI). Current guidelines suggest enhanced surveillance and streptococcal isolate storage after a single case of iGAS, as well as a full epidemiological investigation that includes screening health care workers (HCWs) from several sites after 2 cases. Current guidelines do not recommend routine screening of household members of a patient with iGAS. METHODS: We conducted studies of 3 patients with iGAS puerperal sepsis and related epidemiologic and molecular investigations. RESULTS: Identical GAS emm gene types were found in pharyngeal cultures of 3 asymptomatic spouses of patients with iGAS puerperal sepsis. HCWs screened negative for GAS, and emm typing indicated that other iGAS cases from this hospital were sporadic and not related to the puerperal cases. CONCLUSIONS: The concurrent presence of the same emm type in a household member practically excludes the option of an inadvertent HAI or facility outbreak. Hence, we suggest that screening close family members for asymptomatic GAS carriage should be performed early as a part of infection prevention measures, as doing so would have significant utility in saving time and resources related to a full epidemiological inquiry.

Long-Chain Polyunsaturated Fatty Acids in the Green Microalga Lobosphaera incisa Contribute to Tolerance to Abiotic Stresses.

Lobosphaera incisa is a green microalga that accumulates high levels of the valuable omega-6 long-chain polyunsaturated fatty acids (LC-PUFA) arachidonic acid (ARA, 20:4n-6) in triacylglycerols (TAG) under nitrogen (N) starvation. LC-PUFA accumulation is a rare trait in photosynthetic microalgae with insufficiently understood physiological significance. In this study, RNAi was attempted, for the first time in L. incisa, to produce knockdown lines for the Δ5 desaturase gene. Two lines, termed modified lines, which were isolated during screening for transgenic events, demonstrated alterations in their LC-PUFA profile, ARA-biosynthesis gene expression and lipid class distribution. In line M5-78, which appeared to carry a mutation in the Δ6 elongase gene, LC-PUFA were substituted by 18:3n-6 in all glycerolipids. Line M2-35, for which the exact genetic background has not been established, displayed a dramatic reduction in 20:4n-6, concomitant with an augmented proportion of 18:1n-9, in particular in the extraplastidial membrane lipids and TAG. The physiological responses of the modified lines to stressful conditions were compared with the wild type and the Δ5 desaturase mutant. In the N-replete cells of modified lines, the frequency of lipid droplets was reduced, while a number of starch grains increased, suggesting altered partitioning of assimilated carbon into reserve products. Furthermore, both lines exhibited reduced ability to accumulate TAG under N deprivation and recover from N starvation. Both lines demonstrated lower photosynthetic pigment contents, impairments in photosynthesis under a range of stressful conditions, and less efficient functioning of photoprotection under optimal conditions. Possible implications of fatty acids modifications in the stress response of L. incisa are addressed.

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress.

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n-6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25°C; chilling temperatures (≤15°C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n-6). In nutrient-replete cells grown at 25°C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n-3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15°C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might 'lock' violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.

A prospective survey of Pseudomonas aeruginosa colonization and infection in the intensive care unit.

BACKGROUND: Pseudomonas aeruginosa (PA) surveillance may improve empiric antimicrobial therapy, since colonizing strains frequently cause infections. This colonization may be 'endogenous' or 'exogenous', and the source determines infection control measures. We prospectively investigated the sources of PA, the clinical impact of PA colonization upon admission and the dynamics of colonization at different body sites throughout the intensive care unit stay. METHODS: Intensive care patients were screened on admission and weekly from the pharynx, endotracheal aspirate, rectum and urine. Molecular typing was performed using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain reaction (ERIC-PCR). RESULTS: Between November 2014 and January 2015, 34 patients were included. Thirteen (38%) were colonized on admission, and were at a higher risk for PA-related clinical infection (Hazard Ratio = 14.6, p = 0.0002). Strains were often patient-specific, site-specific and site-persistent. Sixteen out of 17 (94%) clinical isolates were identical to strains found concurrently or previously on screening cultures from the same patient, and none were unique. Ventilator associated pneumonia-related strains were identical to endotracheal aspirates and pharynx screening (87-75% of cases). No clinical case was found among patients with repeated negative screening. CONCLUSION: PA origin in this non-outbreak setting was mainly 'endogenous' and PA-strains were generally patient- and site-specific, especially in the gastrointestinal tract. While prediction of ventilator associated pneumonia-related PA-strain by screening was fair, the negative predictive value of screening was very high.

Water faucets as a source of Pseudomonas aeruginosa infection and colonization in neonatal and adult intensive care unit patients.

We investigated the occurrence of Pseudomonas aeruginosa in our neonatal and adult intensive care units. Using enterobacterial repetitive intergenic consensus polymerase chain reaction, we showed spatial and temporal associations with clonal identity between patients' and adjacent faucets' clones. Both units' taps were highly colonized with P aeruginosa and with other waterborne bacteria. In the neonatal intensive care unit, strict use of sterile water for bathing neonates may have contributed to a reduction in clinical isolation of P aeruginosa postintervention.

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.

Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations.

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.

Growth, lipid production and metabolic adjustments in the euryhaline eustigmatophyte Nannochloropsis oceanica CCALA 804 in response to osmotic downshift.

We investigated the effects of osmotic downshift induced by the transfer of Nannochloropsis oceanica CCALA 804 from artificial seawater medium (27 g L(-1) NaCl) to the same medium without NaCl or freshwater modified BG-11 medium (mBG-11) as a function of photosynthetically active radiation (170, 350, or 700 µmol photon m(-2) s(-1)). Alterations in growth, total fatty acid (FA) content and FA composition of individual lipid classes, and in relative contents of metabolites relevant to osmotic adjustments were studied. Cells displayed remarkable tolerance to the osmotic downshift apart from some swelling, with no substantial lag or decline in cell division rate. Biomass accumulation and chlorophyll a content were enhanced upon downshifting, especially under the highest irradiance. The highest chlorophyll a and eicosapentaenoic acid (EPA) biomass and culture contents were determined in the cultures grown in mBG-11. Two days after transfer to 0 g L(-1) NaCl, the proportion in total acyl lipids of the major chloroplast galactolipid monogalactosyldiacylglycerol, a major depot of EPA, increased twofold, along with a modest change in the proportion of digalactosyldiacylglycerol (DGDG). EPA percentage decreased in DGDG and increased in the extraplastidial lipid phosphatidylethanolamine. Metabolite profiling by GC-MS analysis revealed a sharp decrease in metabolites potentially involved in osmoregulation, such as mannitol and proline, while proline-cycle intermediates and some free sugars increased. The stress-induced polyamine spermidine decreased ca. one order of magnitude, while its catabolic product-the non-protein amino acid γ-amino butyric acid-increased twofold, as did the stress-related sugars trehalose and talose. Biochemical mechanisms governing osmotic plasticity and implications for optimization of EPA production by N. oceanica CCALA 804 under variable cultivation conditions are discussed.