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Background Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with sporadic outbreaks in Cameroon since 2006. Viral whole genomes were generated to analyze the origins of evolutionary lineages, the potential of emergence/re-emergence, and to infer transmission dynamics of recent Cameroon CHIKV outbreak strains. Methods Samples collected between 2016 and 2019 during CHIKV outbreaks in Cameroon were screened for CHIKV using reverse transcription PCR (RT-PCR), followed by whole genome sequencing of positive samples. Results Three coding-complete CHIKV genomes were obtained from samples, which belong to an emerging sub-lineage of the East/Central/South African genotype and formed a monophyletic taxon with previous Central African strains. This clade, which we have named the new Central African clade, appears to be evolving at 3.0 × 10−4 nucleotide substitutions per site per year (95% highest posterior density (HPD) interval of 1.94 × 10−4 to 4.1 × 10−4). Notably, mutations in the envelope proteins (E1-A226V, E2-L210Q, and E2-I211T), which are known to enhance CHIKV adaptability and infectious potential in Aedes albopictus, were present in all strains and mapped to established high-density Ae. albopictus populations. Conclusions These new CHIKV strains constitute a conserved genomic pool of an emerging sub-lineage, reflecting a putative vector host adaptation to Ae. albopictus, which has practically displaced Aedes aegypti from select regions of Cameroon.

Background: Here we compare the performance of the high-throughput BD COR System (COR) to the Viper LT System (Viper) using the BD Onclarity HPV assay. Research Design and Methods: Remnant clinical specimens, contrived specimens in SurePath (BD) and PreservCyt (Hologic) media, and prospective clinical specimens in BD Cervical Brush Diluent (CBD) were tested. Outcomes included intra-laboratory agreement of Onclarity results on COR and inter-system agreement between COR and Viper. Results: Onclarity reproducibility on COR resulted in standard deviation and correlation of variation of Ct values ranging from 0.14 to 1.98 and 0.49% to 2.15%, respectively, for contrived specimens, and 0.9–3.08 and 2.89–9.21%, respectively, for clinical specimens. In the COR and Viper clinical agreement study, OPA for Onclarity ranged from 97.1%-98.9%, depending on the collection media type. PPA values for pooled, HPV(+) specimens at low positive (C95), and moderate positive (3XC95) target concentrations were ≥95.0% and 100%, respectively; PPA values associated with HPV 16, 18, 31, 45, 33/58, 52, 35/39/68, 51, and 56/59/66, individually, ranged from 93.8%-100%. Conclusions: Onclarity performance on COR is equivalent to Viper, and is accurate and reproducible for detection of all high-risk HPV genotypes, with a throughput of 330 results from a single 8-hour shift.

Author(s): Schooley, Robert T; Biswas, Biswajit; Gill, Jason J; Hernandez-Morales, Adriana; Lancaster, Jacob; Lessor, Lauren; Barr, Jeremy J; Reed, Sharon L; Rohwer, Forest; Benler, Sean; Segall, Anca M; Taplitz, Randy; Smith, Davey M; Kerr, Kim; Kumaraswamy, Monika; Nizet, Victor; Lin, Leo; McCauley, Melanie D; Strathdee, Steffanie A; Benson, Constance A; Pope, Robert K; Leroux, Brian M; Picel, Andrew C; Mateczun, Alfred J; Cilwa, Katherine E; Regeimbal, James M; Estrella, Luis A; Wolfe, David M; Henry, Matthew S; Quinones, Javier; Salka, Scott; Bishop-Lilly, Kimberly A; Young, Ry; Hamilton, Theron | Abstract: Volume 61, no. 10, e00954-17, 2017, https://doi.org/10.1128/AAC.00954-17. Materials and Methods, 2nd paragraph, 2nd sentence: “Phages provided by the Biological Defense Research Directorate (BDRD) of the Naval Medical Research Center (AB-Navy1, AB-Navy4, AB-Navy71, AB-Navy97, and AbTP31) were isolated from various environmental samples by using routine isolation techniques, as previously described (9)” should read “Phages provided by the Biological Defense Research Directorate (BDRD) of the Naval Medical Research Center (AB-Navy1, AB-Navy4, AB-Navy97, and AbTP31) were isolated from various environmental samples by using routine isolation techniques, as previously described (9), except for one phage (AB-Navy71, also known as phage vB_Ab-M-G7, DSM strain no. 25639), which was purchased from Liebniz Institute DSMZ (Germany).”

Staphylococcus aureus (S. aureus) is a common cause of surgical site infections (SSIs) globally. Data on the occurrence of methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) among patients with surgical site infections (SSIs) in sub-Saharan African are scarce. We characterized S. aureus from SSIs in Ghana using molecular methods and antimicrobial susceptibility testing (AST). Wound swabs or aspirate samples were collected from subjects with SSIs. S. aureus was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), AST was performed by Kirby-Bauer disk diffusion, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Detection of spa, mecA, and pvl genes was performed by polymerase chain reaction (PCR). Whole-genome sequencing (WGS) was done using the Illumina MiSeq platform. Samples were collected from 112 subjects, with 13 S. aureus isolates recovered. Of these, 92% were sensitive to co-trimoxazole, 77% to clindamycin, and 54% to erythromycin. Multi-drug resistance was detected in 5 (38%) isolates. The four mecA gene-positive MRSA isolates detected belonged to ST152 (n = 3) and ST5 (n = 1). In total, 62% of the isolates were positive for the Panton-Valentine leukocidin (pvl) toxin gene. This study reports, for the first time, a pvl-positive ST152-t355 MRSA clone from SSIs in Ghana. The occurrence of multi-drug-resistant S. aureus epidemic clones suggests that continuous surveillance is required to monitor the spread and resistance trends of S. aureus in hospital settings in the country.

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, includingAcinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDRA. baumanniiinfection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for anA. baumanniiisolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of theA. baumanniiinfection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Here, we report a complex case that involved a pediatric patient who experienced recalcitrant multidrug-resistant Pseudomonas aeruginosa infection complicated by bacteremia/sepsis; our antibacterial options were limited because of resistance, allergies, and suboptimal source control. A cocktail of 2 bacteriophages targeting the infectious organism introduced on 2 separate occasions sterilized the bacteremia.

1H nuclear magnetic resonance (1H NMR)-based metabolomics was utilized to elucidate the earthworm sub-lethal toxicity after exposure to the persistent environmental contaminant phenanthrene. Earthworms were exposed to 0.05, 0.2 and 0.4 mg/cm2 of phenanthrene [which correspond to 1/32nd to 1/4th of the 48-h LC50 (concentration that causes 50 % mortality), respectively] via contact tests over 1, 2 and 3 days of dermal contact. 1H NMR-based metabolomic analysis of the polar and non-polar fractions of the earthworm tissue extracts revealed heightened Eisenia fetida toxic responses with both longer exposure times and higher phenanthrene concentrations. Principal component analysis (PCA) of the polar fraction showed significant separation between control and exposed earthworms along PC1 for all phenanthrene concentrations on each day. The PCA of the non-polar fraction showed significant separation between the controls and exposed earthworms for only the first day of exposure. These results suggested that alanine, glutamate, maltose, and fatty acids were potential indicators of phenanthrene exposure. Interruption in energy production due to a deactivation of the succinate dehydrogenase enzyme in the Krebs cycle was also postulated in exposed earthworms. Cross-validated partial least squares-regression models showed that the polar metabolic profile of E. fetida was weakly but significantly correlated to phenanthrene exposure concentrations after day 1 and day 2 of exposure. Overall, this study indicates that with longer exposures, contact time becomes more important than concentration in discriminating between control and exposed earthworms. This study also shows that NMR-based metabolomics has promise as a powerful ecotoxicological tool for elucidating the mode of toxicity of contaminants.

1 H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm 2 of phenanthrene (1/64th of the LC 50 ) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by 1 H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies.

Eisenia fetida earthworms were exposed to phenanthrene for thirty days to compare hydroxypropyl-beta-cyclodextrin (HPCD) extraction of soil and 1H NMR earthworm metabolomics as indicators of bioavailability. The phenanthrene 28-d LC50 value was 750 mg/kg (632-891, 95% confidence intervals) for the peat soil tested. The initial phenanthrene concentration was 319 mg/kg, which biodegraded to 16 mg/kg within 15 days, at which time HPCD extraction suggested that phenanthrene was no longer bioavailable. Multivariate statistical analysis of 1H NMR spectra for E. fetida tissue extracts indicated that phenanthrene exposed and control earthworms differed throughout the 30 day experiment despite the low phenanthrene concentrations present after 15 days. This metabolic response was better correlated to total phenanthrene concentrations (Q2 = 0.59) than HPCD-extractable phenanthrene concentrations (Q2 = 0.46) suggesting that 1H NMR metabolomics offers considerable promise as a novel, molecular-level method to directly monitor the bioavailability of contaminants to earthworms in the environment.

The existence of two motive forces on a Crookes radiometer has complicated the investigation of either force independently. The thermal creep shear force in particular has been subject to differing interpretations of the direction in which it acts and its order of magnitude. In this article we provide a horizontal vane radiometer design which isolates the thermal creep shear force. The horizontal vane radiometer is explored through experiment, kinetic theory, and the Direct Simulation Monte Carlo (DSMC) method. The qualitative agreement between the three methods of investigation is good expect for a dependence of the force on the width of the vane even when the temperature gradient is narrower than the vane which is present in the DSMC method results but not in the theory. The experimental results qualitatively resemble the theory in this regard. The quantitative agreement between the three methods of investigation is better than an order of magnitude in the cases examined. The theory is closer to the experimental values for narrow vanes and the simulations are closer to the experimental values for the wide vanes. We find that the thermal creep force acts from the hot side to the cold side of the vane. We also find the peak in the radiometer's angular speed as a function of pressure is explained as much by the behavior of the drag force as by the behavior of the thermal creep force.

Extensive population-level genetic variability at the Salmonella rfb locus, which encodes enzymes responsible for synthesis of the O-antigen polysaccharide, is thought to have arisen through frequency-dependent selection (FDS) by means of exposure of this pathogen to host immune systems. The FDS hypothesis works well for pathogens such as Haemophilus influenzae and Neisseria meningitis , which alter the composition of their O-antigens during the course of bloodborne infections. In contrast, Salmonella remains resident in epithelial cells or macrophages during infection and does not have phase variability in its O-antigen. More importantly, Salmonella shows host–serovar specificity, whereby strains bearing certain O-antigens cause disease primarily in specific hosts; this behavior is inconsistent with FDS providing selection for the origin or maintenance of extensive polymorphism at the rfb locus. Alternatively, selective pressure may originate from the host intestinal environment itself, wherein diversifying selection mediated by protozoan predation allows for the continued existence of Salmonella able to avoid consumption by host-specific protozoa. This selective pressure would result in high population-level diversity at the Salmonella rfb locus without phase variation. We show here that intestinal protozoa recognize antigenically diverse Salmonella with different efficiencies and demonstrate that differences solely in the O-antigen are sufficient to allow for prey discrimination. Combined with observations of the differential distributions of both serotypes of bacterial species and their protozoan predators among environments, our data provides a framework for the evolution of high genetic diversity at the rfb locus and host-specific pathogenicity in Salmonella .

Nuclear magnetic resonance (NMR) – based metabolomics has the potential to identify toxic responses of contaminants within a mixture in contaminated soil. This study evaluated the metabolic response of Eisenia fetida after exposure to an array of organic compounds to determine whether contaminant-specific responses could be identified. The compounds investigated in contact tests included: two pesticides (carbaryl and chlorpyrifos), three pharmaceuticals (carbamazephine, estrone and caffeine), two persistent organohalogens (Aroclor 1254 and PBDE 209) and two industrial compounds (nonylphenol and dimethyl phthalate). Control and contaminant-exposed metabolic profiles were distinguished using principal component analysis and potential contaminant-specific biomarkers of exposure were found for several contaminants. These results suggest that NMR-based metabolomics offers considerable promise for differentiating between the different toxic modes of action (MOA) associated with sub-lethal toxicity to earthworms.

The nitrogen regulatory protein P II and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P II homolog, GlnJ, has higher affinity for an AmtB 1 -containing membrane than the other two P II homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB 1 complex in the presence of ATP and 2-KG, supporting a role for P II as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K d of 2-KG for GlnJ bound to AmtB 1 is 340 μM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 μM. This supports a model where multiple 2-KG and ATP molecules must bind a P II trimer to stimulate release of P II from AmtB 1 , in contrast to the lower 2-KG requirement for productive uridylylation of P II by GlnD.

The AmtB protein transports uncharged NH3 into the cell, but it also interacts with the nitrogen regulatory protein PII, which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three PII homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2, have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB1 are defective in their response to both addition and darkness, while mutants lacking AmtB2 show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB1-PII regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by PII is proposed. Other results demonstrate a degree of specificity in interaction of AmtB1 with the different PII homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which PII proteins regulate processes involved in nitrogen acquisition and utilization.

The C 18 -carboxypropylbetaine (CB-18) procedure for processing respiratory specimens for the detection of mycobacteria was shown to provide significant increases in sensitivity by smear and culture. However, the procedure also produced increased contamination, a loss in liquid culture sensitivity, and a reduction in smear specificity. Because of these observations, the toxicity of CB-18 and the nature of the contamination were characterized. Preincubation in 1 mM CB-18 impacted viability in a time-dependent fashion, but the magnitude of the loss was species and isolate dependent. Mycobacterium tuberculosis isolates were the most susceptible, losing 20 to 30% of the CFU within 30 min and 30 to 60% after 3 h, whereas Mycobacterium avium and Mycobacterium fortuitum isolates were unaffected by CB-18. In liquid culture, when the concentration of CB-18 exceeded 5 μg/ml, there was an impact on growth characteristics for the most susceptible M. tuberculosis isolate. In contrast, M. fortuitum isolates were able to grow in 100 μg of CB-18 per ml. In liquid culture, the deleterious effects of CB-18 were enhanced in the presence of antibiotics, whereas growth on solid media was not similarly affected. Supplementation of the resuspension buffer with 0.15% lecithin alleviated toxicity. Initial attempts to modify the CB-18 procedure to control contamination incorporated acids or alkalis; however, losses in culture sensitivity occurred. Studies to identify these contaminants led to the development of a sediment resuspension buffer that contained lytic enzymes to combat contamination and lecithin to alleviate toxicity. This formulation included lysozyme, zymolyase, and Cytophaga and Trichoderma extracts and was seen to reduce contamination to acceptable levels (

Environmental contextPhenanthrene is a persistent soil contaminant, whose toxic mode of action in earthworms has not been fully examined. We adopt a metabolomics approach, using 1H nuclear magnetic resonance (NMR) spectroscopy, to measure the response of earthworms to sub-lethal phenanthrene exposure. The results indicate that NMR-based metabolomics may be used to monitor responses to sub-lethal levels of contaminants and to delineate their toxic mode of action. Abstract1H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida to sub-lethal phenanthrene exposure. E. fetida were exposed via contact tests to six sub-lethal (below the measured LC50 of 1.6 mg cm–2) concentrations of phenanthrene (0.8–0.025 mg cm–2) for 48 h. Multivariate statistical analysis of the 1H NMR spectra of earthworm tissue extracts revealed a two-phased mode of action (MOA). At exposures below 1/16th of the LC50, the MOA was characterised by a linear correlation between the metabolic response and exposure concentration. At exposures ≥1/16th of the LC50, the metabolic response to phenanthrene appeared to plateau, indicating a distinct change in the MOA. Further data analysis suggested that alanine, lysine, arginine, isoleucine, maltose, ATP and betaine may be potential indicators for sub-lethal phenanthrene exposure. Metabolite variation was also found to be proportional to the exposure concentration suggesting that NMR-based earthworm metabolomics is capable of elucidating concentration-dependent relationships in addition to elucidating the MOA of sub-lethal contaminant-exposure.