Population consequences of mutational events: effects of antibiotic resistance on the <Emphasis Type="Italic">r</Emphasis>/<Emphasis Type="Italic">K</Emphasis> trade-off

What are the effects of a mutational event on population dynamics? This eco-evolutionary question has relevance not only to basic biological theories but also to conservation applications. We evaluated the relationship between maximum population growth rate (r _max) and carrying capacity (K) among strains of the bacterium Pseudomonas fluorescens. Each of 65 strains differed from their common ancestor by one naturally acquired phenotypic change conferring antibiotic resistance, brought about by a single mutational event, and each was grown in isolation in four environments. We found no evidence of a trade-off between r _max and K. Rather, strains with rapid growth rates also had high carrying capacity, with little interaction between strain and environment. We conclude that the extensive variation in overall fitness resulting from single mutational events likely masks whatever population trade-offs may exist.     

Analysis of genomic region spanning <Emphasis Type="Italic">Saltol</Emphasis> using SSR markers in rice genotypes showing differential seedlings stage salt tolerance

Micro satellite markers located in the Saltol QTL of 5 Mb region (10.4–15.6 Mb) in chromosome 1 confering seedling stage salt tolerance were used to evaluate 94 rice genotypes. Out of 21, eight SSR markers at Saltol region of Chromosome were found polymorphic. Based on the phenotypic screening, 94 genotypes were grouped as highly tolerant (20), tolerant (18) moderately tolerant (32), sensitive (19) and highly sensitive (5). The marker RM3412 appears to be diagnostic of salinity tolerance and associate to salinity tolerance at seedling stage as it is closely linked to SKC gene. Based on Saltol markers study, CSR 31, CSR 38, CSR 41, CSR 32, Wild 11, CSR 18, Azgo, Pant Dhan 4, Trichi 1, CSR 10 and IR64426-4B-11-1 could not be identified as tolerant genotypes though had expressed tolerant to highly tolerant phenotype to salinity stress at seedling stage, suggesting that QTLs other than Saltol might be controlling their salinity tolerance. It is suggested that these genotypes could serve as potentially novel germplasm and could be exploited for the development of new breeding lines with high level of salinity tolerance by pyramiding of the Saltol and other QTLs.     

Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number

Background

SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI ⁺] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI ⁺] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP ⁺]. However, even though many phenotypic and molecular level features of [ISP ⁺] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.

Results

Here we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp^?) and non-suppressor (Isp⁺) phenotypes. We report that the reason for the difference between the Isp⁺ and the Isp^? phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.

Conclusions

(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.

     

<Emphasis Type="Italic">chr</Emphasis> genes from adaptive replicons are responsible for chromate resistance by <Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400

The chromate ion transporter (CHR) superfamily includes proteins that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 encodes six CHR homologues in its multireplicon genome and has been reported as highly chromate-resistant. The objective of this work was to analyze the involvement of chr redundant genes in chromate resistance by LB400. It was found that B. xenovorans plant rhizosphere strains lacking the megaplasmid are chromate-sensitive, suggesting that the chr gene present in this replicon is responsible for the chromate-resistance phenotype of the LB400 strain. Transformation of a chromate-sensitive B. xenovorans strain with each of the six cloned LB400 chr genes showed that genes from ‘adaptive replicons’ (chrA1b and chr1NCb from chromosome 2 and chrA2 from the megaplasmid) conferred higher chromate resistance levels than chr genes from ‘central’ chromosome 1 (chrA1a, chrA6, and chr1NCa). An LB400 insertion mutant affected in the chrA2 gene displayed a chromate-sensitive phenotype, which was fully reverted by transferring the chrA2 wild-type gene, and partially reverted by chrA1b or chr1NCb genes. These data indicate that chr genes from adaptive replicons, mainly chrA2 from the megaplasmid, are responsible for the B. xenovorans LB400 chromate-resistance phenotype.     

Metal tolerance assisted antibiotic susceptibility profiling in <Emphasis Type="Italic">Comamonas acidovorans</Emphasis>

Metal ions are known selective agents for antibiotic resistance and frequently accumulate in natural environments due to the anthropogenic activities. However, the action of metals that cause the antibiotic resistance is not known for all bacteria. The present work is aimed to investigate the co-selection of metals and antibiotic resistance in Comamonas acidovorans. Tolerance profile of 16 metals revealed that the strain could tolerate high concentrations of toxic metals i.e., Cr (710 ppm), As (380 ppm), Cd (320 ppm), Pb (305 ppm) and Hg (205 ppm). Additionally, metal tolerant phenotypes were subjected to antibiotic resistance profiling; wherein several metal tolerant phenotypes (Cr 1.35-fold; Co-1.33 fold; Mn-1.29 fold) were resistant, while other metal tolerant phenotypes (Mg 1.32-fold; Hg 1.29-fold; Cu 1.28-fold) were susceptible than control phenotype. Metal accumulation may alter the metabolism of C. acidovorans that activates or inactivates the genes responsible for antibiotic resistance, resulting in the resistance and/or susceptibility pattern observed in metal resistant phenotypes.     

Antifungal Susceptibility Testing of <Emphasis Type="Italic">Candida</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis> Species and Mechanisms of Resistance: Implications for Clinical Laboratories

Purpose of Review

Resistance to antifungal drugs amongst Candida species is a growing concern, and azole resistance may be emerging in Cryptococcus species. This review provides a contemporary perspective, relevant to the clinical mycology laboratory, of antifungal susceptibility testing of these fungi, focussing on the challenges of phenotypic and genotypic methodologies to detect drug resistance.

Recent Findings

Standardised CLSI and EUCAST broth microdilution (BMD) susceptibility testing methods are the benchmark to determine clinical breakpoints (CBPs) and/or epidemiological cut-off values (ECVs) MICs for Candida and Cryptococcus spp. Commercial methods may be used but caution is required when employing BMD CBPs/ECVs to interpret results. Species-specific CBPs/ECVs for Candida spp. generally correlate well with predicting likelihood of therapeutic failure or of presence of a drug resistance mechanism with the exception of the echinocandins where the presence of specific FKS gene mutations and not the MIC correlates most accurately with clinical outcome. The relationship of presence of one or more mechanisms of azole resistance and drug MICs is uncertain. Next generation sequencing technology is offering insights into the relationships between susceptibility results obtained by phenotypic and genotypic methods. For Cryptococcus spp., CBPs are not established but species- and genetic type-specific EVCs are useful for guiding therapy where clinically indicated. Isolates of genotype VGII appear to exhibit the highest MICs.

Summary

Antifungal susceptibility testing of yeasts is important to detect drug resistance. For Candida spp., MICs have clinical utility for the azoles but detecting echinocandin resistance by genotypic methods is preferred. For Cryptococcus spp., ECVs are useful in guiding therapy.

     

Screening of rice landraces (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for seedling stage salinity tolerance using morpho-physiological and molecular markers

Traditional rice landraces of coastal area in Bangladesh are distinct regarding their phenotype, response to salt stress and yield attributes. With characterization of these landraces, suitable candidate genes for salinity tolerance could be identified to introgress into modern rice varieties. Therefore, the aim of this experiment was to uncover prospective rice landraces tolerant to salinity. Relying on morphological, biochemical and molecular parameters 25 rice genotypes were tested for salt tolerance at germination and seedling stage. At germination stage 0 and 12 dSm^?1 salinity were imposed on rice genotypes. Ward’s cluster analysis divided rice genotypes into three clusters (susceptible, moderately tolerant and tolerant) based on the physiological indices. The tolerant rice landraces to salinity were Sona Toly, Nakraji and Komol Bhog. At seedling stage screening was performed following IRRI standard protocol at 12 dSm^?1 salinity level. Based on all morphological and biochemical parameters Komol Bhog was identified as the highly salinity tolerant landrace while Bolonga, Sona Toly, Dud Sail, Tal Mugur and Nakraji were found as tolerant to salinity. Molecular characterization using two simple sequence repeats (SSR) markers, viz. RM121 and RM337 displayed Bolonga, Til Kapor, Panbra, Sona Toly, Bina Sail, Komol Bhog, Nakraji, Tilkapur, Gajor Goria and Gota were tolerant landraces through genetic similarity in dendrogram. These identified salt-resistant landraces can be used as promising germplasm resources for breeding salt-tolerant high-yielding rice varieties in future.     

Design of a CRISPR-Cas system to increase resistance of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> to bacteriophage SPP1

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.     

Genomic regions controlling components of resistance for pea rust caused by Uromyces fabae (Pers.) de-Bary

Pea rust caused by Uromyces fabae (Pers.) de-Bary is an important disease in subtropical regions of the world. The use of partial resistance or slow rusting is an important strategy for developing varieties having durable rust resistance. A mapping population of 136 F_6:7 Recombinant Inbred Lines (RILs) derived from the cross HUVP 1?×?FC 1 was evaluated for disease severity percent (DS%) and three components of slow rusting, number of aecial pustules per leaf (AP), leaf area covered by sporulating pustules (LASP) and number of aecial cups per leaf (TNAC) during crop seasons 2006–07 and 2007–08 in polyhouse and field experiments. The components were governed by four quantitative trait loci, two major (Qruf on LGVII, Qruf2 on LGI), and two minor QTLs (Qruf1 on LG VII and Qruf3 on LGVI). This confirmed the positions of one each of the major (Qruf) and minor (Qruf1) QTLs and also detected two new QTLs Qruf2 and Qruf3. The new major QTL Qruf2 (phenotypic variance 21.3 to 29.6 %) appeared to be the most important component-specific QTL and played key role in deciding disease resistance. The minor QTL Qruf3 appeared environment-specific and contributed by the susceptible parent.     

Assessment of genetic diversity and structure in cocoa trees (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Côte d’Ivoire with reference to their susceptibility to Cocoa swollen shoot virus disease (CSSVD)

Resistance to Cocoa Swollen Shoot Virus disease (CSSVD) is becoming an increasingly important criterion for selection of new cocoa cultivars in Côte-d’Ivoire, where the disease resurfaced since 2003. This virus can seriously affect the yield of trees with a loss of 25%, 1 year after infection, to around 100% 3 years after. In order to find tolerant plant material, 337 farm accessions have been collected on fields affected by CSSVD, according to the status of accessions potentially tolerant (APT) or susceptible (APS). Both phenotypic groups were genotyped using 30 microsatellite markers (SSR) in the presence of representative clones of the ten genetic groups of cocoa. This study revealed 214 alleles with the set of primer pairs used. The number of alleles per locus was between 3 and 16 with an average of 7.13 alleles per locus. The results showed a high contribution of genetic diversity within population (H_s?=?0.51) to the total genetic diversity (H_t?=?0.53) for the two studied groups. There was no significant difference between tolerant and susceptible groups (Fst?=?0.05). These results suggest that APT could be a potential genetic reservoir for other traits of interest associated with virus resistance. The phylogenetic tree, as the STRUCTURE analysis of Ivorian cocoa population, showed a distribution of individuals following four groups marked by a high contribution of group 4 (Nanay, Maranon, Guiana) followed by group 2 (Criollo), and group 1 (Amelonado), and a lower contribution of group 3 (Iquitos, Purus, Nacional, Curaray, Contamana).     

Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.     

The role of memory in non-genetic inheritance and its impact on cancer treatment resistance

Intra-tumour heterogeneity is a leading cause of treatment failure and disease progression in cancer. While genetic mutations have long been accepted as a primary mechanism of generating this heterogeneity, the role of phenotypic plasticity is becoming increasingly apparent as a driver of intra-tumour heterogeneity. Consequently, understanding the role of this plasticity in treatment resistance and failure is a key component of improving cancer therapy. We develop a mathematical model of stochastic phenotype switching that tracks the evolution of drug-sensitive and drug-tolerant subpopulations to clarify the role of phenotype switching on population growth rates and tumour persistence. By including cytotoxic therapy in the model, we show that, depending on the strategy of the drug-tolerant subpopulation, stochastic phenotype switching can lead to either transient or permanent drug resistance. We study the role of phenotypic heterogeneity in a drug-resistant, genetically homogeneous population of non-small cell lung cancer cells to derive a rational treatment schedule that drives population extinction and avoids competitive release of the drug-tolerant sub-population. This model-informed therapeutic schedule results in increased treatment efficacy when compared against periodic therapy, and, most importantly, sustained tumour decay without the development of resistance.     

A Highly Promiscuous Integron,Plasmids, Extended Spectrum Beta Lactamases and Efflux Pumps as Factors Governing Multidrug Resistance in a Highly Drug Resistant <Emphasis Type="Italic">Vibrio fluvialis</Emphasis> Isolate BD146 from Kolkata,India

In an earlier study from this laboratory, Vibrio fluvialis BD146, a clinical isolate from Kolkata, India, 2002, was found to be resistant to all the fourteen antibiotics tested. It harboured a high copy number plasmid pBD146 and a low copy number plasmid. In the present study, a more detailed analysis was carried out to unravel different resistance mechanisms in this isolate. Sequencing showed that variable region of class 1 integron located on low copy number plasmid harbored arr3-cmlA-bla _OXA10-aadA1 gene cassettes. Analysis for extended spectrum beta lactamases (ESBLs) revealed that BD146 was ESBL positive. Efflux pumps were involved in the drug resistance phenotype for chloramphenicol, kanamycin, streptomycin and tetracycline. Sequence analysis of pBD146 revealed the presence of genes encoding BDint an integrase with a unique sequence having little similarity to other known integrases, toxin–antitoxin (parE/parD), a replicase, trimethoprim resistance (dfrVI) and quinolone resistance (qnrVC5). Presence of cmlA, putative novel integrase and toxin–antitoxin system in V. fluvialis has been documented for the first time in this report. pBD146 showed 99% sequence similarity with pVN84 from V. cholerae O1 of Vietnam, 2004 and a plasmid from V. parahaemolyticus v110 of Hong Kong, 2010. Conjugation experiments proved the ability of pBD146 and the low copy number plasmid, to get transferred to another host imparting their antibiotic resistance traits to the transconjugants. Therefore, present study has indicated that plasmids played an important role for dissemination of drug resistance.     

Antibiotic resistance of pathogenic <Emphasis Type="Italic">Acinetobacter</Emphasis> species and emerging combination therapy

The increasing antibiotic resistance of Acinetobacter species in both natural and hospital environments has become a serious problem worldwide in recent decades. Because of both intrinsic and acquired antimicrobial resistance (AMR) against last-resort antibiotics such as carbapenems, novel therapeutics are urgently required to treat Acinetobacter-associated infectious diseases. Among the many pathogenic Acinetobacter species, A. baumannii has been reported to be resistant to all classes of antibiotics and contains many AMR genes, such as bla_ADC (Acinetobacter-derived cephalosporinase). The AMR of pathogenic Acinetobacter species is the result of several different mechanisms, including active efflux pumps, mutations in antibiotic targets, antibiotic modification, and low antibiotic membrane permeability. To overcome the limitations of existing drugs, combination theraphy that can increase the activity of antibiotics should be considered in the treatment of Acinetobacter infections. Understanding the molecular mechanisms behind Acinetobacter AMR resistance will provide vital information for drug development and therapeutic strategies using combination treatment. Here, we summarize the classic mechanisms of Acinetobacter AMR, along with newly-discovered genetic AMR factors and currently available antimicrobial adjuvants that can enhance drug efficacy in the treatment of A. baumannii infections.     

Comparison of antibiotic resistance and copper tolerance of <Emphasis Type="Italic">Enterococcus</Emphasis> spp. and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from piglets before and after weaning

In China, antimicrobials and copper are used extensively as growth-promoting agents for piglets. This study aimed to characterize the role of in-feed copper in the emergence of copper-tolerant and antibiotic-resistant Enterococcus and Lactobacillus isolates in Chinese pig farms. Feces of the same eight piglets from four litters at 7 and 55 days old and their mothers were traced in order to isolate Enterococcus spp. and Lactobacillus spp.. The minimum inhibitory concentrations of 10 antimicrobials and copper sulfate were determined using an agar dilution method. The feed levels of Cu²⁺ for lactating sows, suckling piglets, and weaned piglets were 6, 177, and 18 mg/kg, respectively. All the 136 Enterococcus isolates were sensitive to vancomycin; and the resistance rates to penicillin, enrofloxacin, and high level streptomycin resistance increased significantly after weaning. For the 155 Lactobacillus isolates, the resistance rates to ampicillin, chloramphenicol, tetracycline, and enrofloxacin were significantly higher in weaned piglets. The ratios of copper tolerant Enterococcus and Lactobacillus isolates both increased significantly after weaning (P < 0.05). A phenotypic correlation was observed after classifying the isolates into two groups (CuSO₄ MIC₅₀ < 16 or ≧16 for enterococci; CuSO₄ MIC₅₀ < 12 or ≧12 for lactobacilli) and comparing the antimicrobial-resistant percentage of two groups. On species level, a significant increase of E. faecalis to enrofloxacin was observed in line with the increase of copper MIC (P < 0.05). The findings revealed the changes of the antibiotic resistance and copper tolerance level of enterococci and lactobacilli between suckling and weaned piglets and demonstrated that there might be a strong association between in-feed copper and increased antibiotic resistance in enterococci and lactobacilli in Chinese intensive swine farms.     

The response of transgenic <Emphasis Type="Italic">Brassica</Emphasis> species to salt stress: a review

Salt stress is considered one of the main abiotic factors to limit crop growth and productivity by affecting morpho-physiological and biochemical processes. Genetically, a number of salt tolerant Brassica varieties have been developed and introduced, but breeding of such varieties is time consuming. Therefore, current focus is on transgenic technology, which plays an important role in the development of salt tolerant varieties. Various salt tolerant genes have been characterized and incorporated into Brassica. Therefore, such genetic transformation of Brassica species is a significant step for improvement of crops, as well as conferring salt stress resistance qualities to Brassica species. Complete genome sequencing has made the task of genetically transforming Brassica species easier, by identifying desired candidate genes. The present review discusses relevant information about the principles which should be employed to develop transgenic Brassica species, and also will recommend tools for improved tolerance to salinity.     

Genetic mapping of a novel recessive allele for non-glaucousness in wild diploid wheat <Emphasis Type="Italic">Aegilops tauschii</Emphasis>: implications for the evolution of common wheat

Cuticular wax on the aerial surface of plants has a protective function against many environmental stresses. The bluish–whitish appearance of wheat leaves and stems is called glaucousness. Most modern cultivars of polyploid wheat species exhibit the glaucous phenotype, while in a wild wheat progenitor, Ae. tauschii, both glaucous and non-glaucous accessions exist. Iw2, a wax inhibitor locus on the short arm of chromosome 2D, is the main contributor to this phenotypic variation in Ae. tauschii, and the glaucous/non-glaucous phenotype of Ae. tauschii is usually inherited by synthetic hexaploid wheat. However, a few synthetic lines show the glaucous phenotype although the parental Ae. tauschii accessions are non-glaucous. Molecular marker genotypes indicate that the exceptional non-glaucous Ae. tauschii accessions share the same genotype in the Iw2 chromosomal region as glaucous accessions, suggesting that these accessions have a different causal locus for their phenotype. This locus was assigned to the long arm of chromosome 3D using an F₂ mapping population and designated W4, a novel glaucous locus in Ae. tauschii. The dominant W4 allele confers glaucousness, consistent with phenotypic observation of Ae. tauschii accessions and the derived synthetic lines. These results implied that glaucous accessions of Ae. tauschii with the W2W2iw2iw2W4W4 genotype could have been the D-genome donor of common wheat.     

Role of the PTEN protein in the multidrug resistance of prostate cancer cells

At present, there is no doubt that the signal transduction pathway P13K/Akt/PTEN/mTOR, controlled by phosphatidylinositol-3-kinase, is involved in tumor cell resistance to a number of drugs. Another well-known mechanism determining drug resistance in tumors is associated with the activity of drug transporters of the ABC superfamily (first of all, P-glycoprotein (Pgp), MRP1, BCRP, and LRP). Several mechanisms of cell defense can simultaneously operate in one cell. The interplay of different mechanisms involved in drug resistance is poorly understood. The PC3 and DU145 human prostate cell lines were used to show that the PTEN functional status determined the cell resistance to some drugs and that correlated with the levels of MRP1 and BCRP. Pgp was not involved in drug resistance of these cells. Introduction of PTEN into PTEN-deficient PC3 cells, as well as rapamycin treatment, inhibited Akt and mTOR and sensitized cells to doxorubicin and vinblastine. Exogenous PTEN altered the MRP1 and BCRP expression. The results indicate that at least two mechanisms of drug resistance operate in prostate cancer cells: the PI3K/Akt/PTEN/mTOR pathway and an elevated MRP1 expression. The mechanisms are interconnected: PTEN and mTOR signaling is involved in MRP1 and BCRP expression regulation.