Emergence of hyper-resistant Escherichia coli MG1655 derivative strains after applying sub-inhibitory doses of individual constituents of essential oils

The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC''s were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub-inhibitory concentrations should be generally avoided, since it could favor the emergence of hyper-resistant strains. To ascertain the true value of EOs and their ICs in the field of food preservation, further research thus needs to be conducted on the induction of increased transient and stable bacterial resistance via such antimicrobial compounds, as revealed in this study

Astronomical Site Ranking Based on Tropospheric Wind Statistics

We present comprehensive and reliable statistics of high altitude wind speeds and the tropospheric flows at the location of five important astronomical observatories. Statistical analysis exclusively of high altitude winds point to La Palma as the most suitable site for adaptive optics, with a mean value of 22.13 m/s at the 200 mbar pressure level. La Silla is at the bottom of the ranking, with the largest average value 200 mbar wind speed(33.35 m/s). We have found a clear annual periodicity of high altitude winds for the five sites in study. We have also explored the connection of high to low altitude atmospheric winds as a first approach of the linear relationship between the average velocity of the turbulence and high altitude winds (Sarazin & Tokovinin 2001). We may conclude that high and low altitude winds show good linear relationships at the five selected sites. The highest correlation coefficients correspond to Paranal and San Pedro Martir, while La Palma and La Silla show similar high to low altitude wind connection. Mauna Kea shows the smallest degree of correlation, which suggests a weaker linear relationship. Our results support the idea of high altitude winds as a parameter for rank astronomical sites in terms of their suitability for adaptive optics, although we have no evidence for adopting the same linear coefficient at different sites. The final value of this linear coefficient at a particular site could drastically change the interpretation of high altitude wind speeds as a direct parameter for site characterization.Comment: 18 pages, 5 figures. Accepted in MNRA

Whole-genome sequencing and genetic analysis reveal novel stress responses to individual constituents of essential oils in Escherichia coli

Food preservation by the use of essential oils (EOs) is being extensively studied because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT, and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral, and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by several detected SNPs, resulting in a frameshift in MarR and an in-frame GyrB ¿G157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM were correlated with the hyperresistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance

Listeria monocytogenes Inhibits Serotonin Transporter in Human Intestinal Caco-2 Ce

Listeria monocytogenes is a Gram-positive bacterium that can cause a serious infection. Intestinal microorganisms have been demonstrated to contribute to intestinal physiology not only through immunological responses but also by modulating the intestinal serotonergic system. Serotonin (5- HT) is a neuromodulator that is synthesized in the intestinal epithelium and regulates the whole intestinal physiology. The serotonin transporter (SERT), located in enterocytes, controls intestinal 5-HT availability and therefore serotonin’s effects. Infections caused by L. monocytogenes are well described as being due to the invasion of intestinal epithelial cells; however, the effect of L. monocytogenes on the intestinal epithelium remains unknown. The main aim of this work, therefore, was to study the effect of L. monocytogenes on SERT. Caco2/TC7 cell line was used as an enterocyte-like in vitro model, and SERT functional and molecular expression assays were performed. Our results demonstrate that living L. monocytogenes inhibits serotonin uptake by reducing SERT expression at the brush border membrane. However, neither inactivated L. monocytogenes nor soluble metabolites were able to affect SERT. The results also demonstrate that L. monocytogenes yields TLR2 and TLR10 transcriptional changes in intestinal epithelial cells and suggest that TLR10 is potentially involved in the inhibitory effect observed on SERT. Therefore, L. monocytogenes, through TLR10-mediated SERT inhibition, may induce increased intestinal serotonin availability and potentially contributing to intestinal physiological changes and the initiation of the inflammatory response.This work was funded by grants from the Spanish Ministry of Science and Innovation and the European Regional Development Fund (ERDF/FEDER) (BFU2010-18971), Zaragoza University (UZ2014- BIO-03), European Social Found (ESF), and the Aragon Regional Government (B61) and the Foundation for the Study of Inflammatory Bowel Diseases in Aragón (ARAINF 2012/0567). E. Latorre and E. Layunta are PhD student fellows from Aragon Regional Government (B105/11 and B022/13)

VIP induces NF-κB1-nuclear localisation through different signalling pathways in human tumour and non-tumour prostate cells

The nuclear factor kappa B (NF-kappa B) is a powerful activator of angiogenesis, invasion and metastasis. Transactivation and nuclear localisation of NF-kappa B is an index of recurrence in prostate cancer. Vasoactive intestinal peptide (VIP) exerts similar effects in prostate cancer models involving increased expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) which are related to NF-kappa B transactivation. Here we studied differential mechanisms of VIP-induced NF-kappa B transactivation in non-tumour RWPE-1 and tumour LNCaP and PC3 human prostate epithelial cells. Immunofluorescence studies showed that VIP increases translocation of the p50 subunit of NF-kappa B1 to the nucleus, an effect that was inhibited by curcumin. The signalling transduction pathways involved are different depending on cell transformation degree. In control cells (RWPE1), the effect is mediated by protein kinase A (PKA) activation and does not implicate extracellular signal-regulated kinase (ERK) or phosphoinositide 3-kinase (PI3-K) pathways whereas the opposite is true in tumour LNCaP and PC3 cells. Exchange protein directly activated by CAMP (EPAC) pathway is involved in transformed cells but not in control cells. Curcumin blocks the activating effect of VIP on COX-2 promoter/prostaglandin E-2 (PGE(2)) production and VEGF expression and secretion. The study incorporates direct observation on COX-2 promoter and suggests that VIP effect on VEGF may be indirectly mediated by PGE(2) after being synthesised by COX-2, thus amplifying the initial signal. We show that the signalling involved in VIP effects on VEGF is CAMP/PKA in non-tumour cells and cAMP/EPAC/ERK/PI3K in tumour cells which coincides with pathways mediating p50 nuclear translocation. Thus, VIP appears to use different pathways for NF-kappa B1 (p50) transactivation in prostate epithelial cells depending on whether they are transformed or not. Transformed cells depend on pro-survival and pro-proliferative signalling pathways involving ERK, PI3-K and cAMP/EPAC which supports the potential therapeutic value of these targets in prostate cancer

Drop Traffic in Microfluidic Ladder Networks with Fore-Aft Structural Asymmetry

We investigate the dynamics of pairs of drops in microfluidic ladder networks with slanted bypasses, which break the fore-aft structural symmetry. Our analytical results indicate that unlike symmetric ladder networks, structural asymmetry introduced by a single slanted bypass can be used to modulate the relative drop spacing, enabling them to contract, synchronize, expand, or even flip at the ladder exit. Our experiments confirm all these behaviors predicted by theory. Numerical analysis further shows that while ladder networks containing several identical bypasses are limited to nearly linear transformation of input delay between drops, mixed combination of bypasses can cause significant non-linear transformation enabling coding and decoding of input delays.Comment: 4 pages, 5 figure

Liquid flow-focused by a gas: jetting, dripping and recirculation

The liquid cone-jet mode can be produced upon stimulation by a co-flowing gas sheath. Most applications deal with the jet breakup, leading to either of two droplet generation regimes: jetting and dripping. The cone-jet flow pattern is explored by direct axisymmetric VOF numerical simulation; its evolution is studied as the liquid flow-rate is increased around the jetting-dripping transition. As observed in other focused flows such as electrospraying cones upon steady thread emission, the flow displays a strong recirculating pattern within the conical meniscus; it is shown to play a role on the stability of the system, being a precursor to the onset of dripping. Close to the minimum liquid flow rate for steady jetting, the recirculation cell penetrates into the feed tube. Both the jet diameter and the size of the cell are accurately estimated by a simple theoretical model. In addition, the transition from jetting to dripping is numerically analyzed in detail in some illustrative cases, and compared, to good agreement, with a set of experiments.Comment: Submitted to the Physical Review E on December 8th, 200

Cross-country migration linked to people who inject drugs challenges the long-term impact of national HCV elimination programmes

To the Editor: As of 2018, the majority of Western European countries – including Spain – have lifted restrictions to therapy based on disease severity in the context of HCV infections.1 Long overdue, most national elimination programmes now also include access to care for people who inject drugs (PWID), 2 who are at the core of ongoing HCV transmission.3 Macías et al.4 have recently shown in this Journal that high viral cure rates can be achieved in this group, hereby providing evidence that targeting PWID in treatment programmes is worthwhile. However, the extent to which such national efforts can reduce the HCV burden not only depends on the uptake into care and treatment success rates, it is also determined by the relative importance of within-country transmission and virus importation from elsewhere. As the chronic nature of most HCV infections hampers reliably reconstructing contact networks from patient interviews, virus genetic data can be a valuable alternative source of information for elucidating the geographic history of virus lineages (e.g. [5], [6]). Using such data, we have recently shown that for the most prevalent subtype among PWID in Spain (40%, 7), HCV1a, infections often link to infections abroad – in recent years >50% link to Western European countries, mostly European Union (EU) member states – as opposed to other infections ..

Gas Dynamic Virtual Nozzle for Generation of Microscopic Droplet Streams

As shown by Ganan-Calvo and co-workers, a free liquid jet can be compressed in iameter through gas-dynamic forces exerted by a co-flowing gas, obviating the need for a solid nozzle to form a microscopic liquid jet and thereby alleviating the clogging problems that plague conventional droplet sources of small diameter. We describe in this paper a novel form of droplet beam source based on this principle. The source is miniature, robust, dependable, easily fabricated, and eminently suitable for delivery of microscopic liquid droplets, including hydrated biological samples, into vacuum for analysis using vacuum instrumentation. Monodisperse, single file droplet streams are generated by triggering the device with a piezoelectric actuator. The device is essentially immune to clogging