WeFaceNano:a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

Background: Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools. Results: Publicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids. Conclusions: WeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.</p

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhali

Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET)-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly) was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS)-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM). In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility

Disentangling the cause of a catastrophic population decline in a large marine mammal

Considerable uncertainties often surround the causes of long-term changes in population abundance. One striking example is the precipitous decline of southern sea lions (SSL; Otaria flavescens) at the Falkland Islands, from 80 555 pups in the mid 1930s to just 5506 pups in 1965. Despite an increase in SSL abundance over the past two decades, the population has not recovered, with the number of pups born in 2014 (minimum 4443 pups) less than 6% of the 1930s estimate. The order-of-magnitude decline is primarily attributed to commercial sealing in Argentina. Here, we test this established paradigm and alternative hypotheses by assessing (1) commercial sealing at the Falkland Islands, (2) winter migration of SSL from the Falkland Islands to Argentina, (3) whether the number of SSL in Argentina could have sustained the reported level of exploitation, and (4) environmental change. The most parsimonious hypothesis explaining the SSL population decline was environmental change. Specifically, analysis of 160 years of winter sea surface temperatures revealed marked changes, including a period of warming between 1930 and 1950 that was consistent with the period of SSL decline. Sea surface temperature changes likely influenced the distribution or availability of SSL prey and impacted its population dynamics. We suggest that historical harvesting may not always be the “smoking gun” as is often purported. Rather, our conclusions support the growing evidence for bottom-up forcing on the abundance of species at lower trophic levels (e.g., plankton and fish) and resulting impacts on higher trophic levels across a broad range of ecosystems

Health state preferences are equivalent in the United States and Trinidad and Tobago

To derive preference weights in Trinidad and Tobago for Quality of Well-being Scale (QWB) health states in order to calculate QWB scores that can be compared to scores calculated from US-derived preference weights. The comparison was to determine whether the QWB scores from these different preference weights would lead to similar conclusions. We conducted in-person household interviews to elicit preferences for 65 health states using a probability sample of 235 adults from Port of Spain, Chaguanas and San Fernando, Trinidad and Tobago. A regression model with correction for within-person clustering of observations was used to obtain preference weights based on case judgments on a 0 (dead) to 10 (“perfect health”) scale. The independent variables were the components of the QWB entered as indicator (0, 1) variables. One hundred and nineteen (51%) respondents provided ratings. The respondents that provided ratings were demographically no different from those that did not. The QWB response patterns were very similar using Trinidad and US weights. The mean (SD) QWB score was 0.750 (0.130) for female respondents and 0.784 (0.125) for male respondents using Trinidad coefficients (t 2, 233&nbsp;=&nbsp;−2.05, P&nbsp;=&nbsp;0.04) and 0.747 (0.131) for female respondents and 0.783 (0.126) for male respondents using US weights (t 2, 233&nbsp;=&nbsp;−2.17, P&nbsp;=&nbsp;0.03). Overall, we found the US and Trinidad and Tobago weights were highly similar and that the choice of either set of weights would lead to similar conclusions

DNA repair and resistence to UV-B radiation in western spotted frogs

assessed DNA repair and resistance to solar radiation in eggs of members of the western spotted frog complex (Rana pretiosa and R. luteiventris), species whose populations are suffering severe range reductions and declines. Specifically, we measured the activity of photoreactivating enzyme (photolyase) in oocytes of spotted frogs. In some species, photoreactivation is the most important mechanism for repair of UV-damaged DNA. Using field experiments, we also compared the hatching success of spotted frog embr yos at natural oviposition sites at three elevations, where some embr yos were subjected to ambient levels of UV-B radiation and others were shielded from UV-B radiation. Compared with other amphibians, photolyase activities in spotted frogs were relatively high. At all sites, hatching success was unaffected by UV-B. Our data support the interpretation that amphibian embr yos with relatively high levels of photolyase are more resistant to UV-B radiation than those with lower levels of photolyase. At the embr yonic stage, UV-B radiation does not presently seem to be contributing to the population declines of spotted frogs.Peer reviewe

Temporal development of the humoral immune response to surface antigens of Moraxella catarrhalis in young infants

The primary Moraxella catarrha/is-specific humoral immune response, and its association with nasopharyngeal colonization, was studied in a cohort of infants from birth to 2 years of age. Results indicated that the levels of antigen-specific IgG, IgA and IgM showed extensive inter-individual variability over time, with IgM and IgA levels to all 9 recombinant domains, from 7 different OMPs, being relatively low throughout the study period. In contrast, the level of antigen-specific IgG was significantly higher for the recombinant domains Hag(385-863), MID764-913, MID962-1200, UspA1(557-704) and UspA2(165-318) in cord blood compared to 6 months of age (P <= 0.001). This was a most likely a consequence of maternal transmission of antigen-specific IgG to newborn babies, possibly indicating a future role for these 3 surface antigens in the development of an effective humoral immune response to M. catarrhalis. Finally, at 2 years of age, the levels of antigen-specific IgG still remained far below that obtained from cord blood samples, indicating that the immune response to M. catarrhalis has not matured at 2 years of age. We provide evidence that a humoral antibody response to OMPs UspA1,UspA2 and Hag/MID may play a role in the immune response to community acquired M. catarrhalis colonization events. (C) 2011 Elsevier Ltd. All rights reserved

In vitro activity of novel apramycin-dextran nanoparticles and free apramycin against selected Dutch and Pakistani Klebsiella pneumonia isolates

Klebsiella pneumoniae are bacteria associated with respiratory tract infections and are increasingly becoming resistant to antibiotics, including carbapenems. Apramycin is a veterinary antibiotic that may have the potential to be re-purposed for use in human health, for example, for the treatment of respiratory tract infections after coupling to inhalable nanoparticles. In the present study, the antibiotic apramycin was formulated with single chain polymeric nanoparticles and tested in free and formulated forms against a set of 13 Klebsiella pneumoniae isolates (from the Netherlands and Pakistan) expressing different aminoglycoside resistance phenotypes. Minimum Inhibitory Concentration, Time Kill Kinetics and biofilm experiments were performed providing evidence for the potential efficacy of apramycin and apramycin-based nanomedicines for the treatment of human Klebsiella pneumonia infections

Do proxies reflect patients' health concerns about urinary incontinence and gait problems?

BACKGROUND: While falls and urinary incontinence are prevalent among older patients, who sometimes rely on proxies to provide their health information, the validity of proxy reports of concern about falls and urinary incontinence remains unknown. METHODS: Telephone interviews with 43 consecutive patients with falls or fear of falling and/or bothersome urinary incontinence and their proxies chosen by patients as most knowledgeable about their health. The questionnaire included items derived from the Medical Outcomes Study Short Form 12 (SF-12), a scale assessing concerns about urinary incontinence (UI), and a measure of fear of falling, the Falls Efficacy Scale (FES). Scores were estimated using items asking the proxy perspective (6 items from the SF-12, 10 items from a UI scale, and all 10 FES items). Proxy and patient scores were compared using intraclass correlation coefficients (ICC, one-way model). Variables associated with absolute agreement between patients and proxies were explored. RESULTS: Patients had a mean age of 81 years (range 75–93) and 67% were female while proxies had a mean age of 70 (range 42–87) and 49% were female. ICCs were 0.63 for the SF-12, 0.52 for the UI scale, and 0.29 for the FES. Proxies tended to understate patients' general health and incontinence concern, but overstate patients' concern about falling. Proxies who lived with patients and those who more often see patients more closely reflected patient FES scores compared to those who lived apart or those who saw patients less often. Internal consistency reliability of proxy responses was 0.62 for the SF-12, 0.86 for the I-QOL, and 0.93 for the FES. In addition, construct validity of the proxy FES scale was supported by greater proxy-perceived fear of falling for patients who received medical care after a fall during the past 12 months (p < .05). CONCLUSION: Caution should be exercised when using proxies as a source of information about older patients' health perceptions. Questions asking about proxies' views yield suboptimal agreement with patient responses. However, proxy scales of UI and fall concern are internally consistent and may provide valid independent information