SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands.

In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmr.753

Survey of the year 2003 commercial optical biosensor literature

Resistance integrons and super‐integrons

Reversing the spread of antibiotic multiresistant bacteria is hampered by ignorance of the natural history of resistance genes, the mobile elements carrying them, and the bacterial hosts harboring them. Using traditional cultivation and cultivation-independent molecular techniques, we quantified antibiotic resistance genes and mobile elements called integrons in poultry house litter from commercial poultry farms. Unexpectedly, the major reservoir for Class 1 integrons in poultry litter is not their previously identified hosts, Gram-negative Enterobacteriaceae such as Escherichia coli. Rather, integrons and associated resistance genes abound in several genera of Gram-positive bacteria that constitute >85% of the litter community compared with Enterobacteriaceae that comprise <2% of this ecosystem. This finding warrants reexamination of our assumptions about the persistence and spread of antibiotic resistance genes.

https://www.pnas.org/content/pnas/101/18/7118.full.pdf

Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter

Over the last fifteen years there have been five pandemics of norovirus (NoV) associated gastroenteritis, and the period of stasis between each pandemic has been progressively shortening. NoV is classified into five genogroups, which can be further classified into 25 or more different human NoV genotypes; however, only one, genogroup II genotype 4 (GII.4), is associated with pandemics. Hence, GII.4 viruses have both a higher frequency in the host population and greater epidemiological fitness. The aim of this study was to investigate if the accuracy and rate of replication are contributing to the increased epidemiological fitness of the GII.4 strains. The replication and mutation rates were determined using in vitro RNA dependent RNA polymerase (RdRp) assays, and rates of evolution were determined by bioinformatics. GII.4 strains were compared to the second most reported genotype, recombinant GII.b/GII.3, the rarely detected GII.3 and GII.7 and as a control, hepatitis C virus (HCV). The predominant GII.4 strains had a higher mutation rate and rate of evolution compared to the less frequently detected GII.b, GII.3 and GII.7 strains. Furthermore, the GII.4 lineage had on average a 1.7-fold higher rate of evolution within the capsid sequence and a greater number of non-synonymous changes compared to other NoVs, supporting the theory that it is undergoing antigenic drift at a faster rate. Interestingly, the non-synonymous mutations for all three NoV genotypes were localised to common structural residues in the capsid, indicating that these sites are likely to be under immune selection. This study supports the hypothesis that the ability of the virus to generate genetic diversity is vital for viral fitness.

/pdf/rapid-evolution-of-pandemic-noroviruses-of-the-gii-4-lineage-3p83g0u5xo.pdf

Rapid evolution of pandemic noroviruses of the GII.4 lineage.

Class 2 integrons (Tn7) were found in all randomly selected epidemic (n = 27) and preepidemic (n = 13) strains of multiresistant Shigella sonnei biotype g. A class 1 integron was also found in two epidemic strains. Gene cassettes within these integrons account for resistance to commonly used therapeutic agents.

Epidemic Strains of Shigella sonnei Biotype g Carrying Integrons

Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.

High-Affinity Aptamers to Subtype 3a Hepatitis C Virus Polymerase Display Genotypic Specificity

Integrons were detected in 37 (72.5%) of 51 Klebsiella spp. producing extended-spectrum beta-lactamases by PCR with primers that targeted integrase genes and cassette regions. PCR and amplicon sequencing of the cassette regions revealed aadB and aadA2 gene cassettes that confer resistance to a range of aminoglycosides. aadB was associated with a class 1 integron on a 28-kb plasmid, pES1, that also contained bla(SHV-12) and IS26.

The aadB Gene Cassette Is Associated with blaSHV Genes in Klebsiella Species Producing Extended-Spectrum β-Lactamases

Integrons have been recognised as important contributors to the acquisition and dissemination of antibiotic resistance in Gram-negative bacteria. In a collection of 19 multi-antibiotic resistant Gram- negative clinical isolates, 47 per cent (9/19) of strains were found to contain one or more integron, using a polymerase chain reaction (PCR) based screening method. Resistance gene cassettes within the integrons were amplified, sequenced and characterised. Antibiotic susceptibility testing demonstrated that resistance phenotypes correlated with the resistance conferred by gene cassettes identified. PCR-screening for integrons and gene cassettes provides a rapid technique for the identification of genetic determinants of resistance in Gram-negative bacteria. Such screening could assist in guiding treatment regimens and complement existing antibiotic resistance surveillance programs by providing information on molecular mechanisms of both resistance and resistance dissemination. (author abstract)

/pdf/polymerase-chain-reaction-screening-for-integrons-can-be-3ifsuyh1na.pdf

Louisa A. Jones

Papers

Epidemic Strains of Shigella sonnei Biotype g Carrying Integrons

High-Affinity Aptamers to Subtype 3a Hepatitis C Virus Polymerase Display Genotypic Specificity

The aadB Gene Cassette Is Associated with blaSHV Genes in Klebsiella Species Producing Extended-Spectrum β-Lactamases

Polymerase chain reaction screening for integrons can be used to complement resistance surveillance programs.