The bacterial ribosome comprises 30 S and 50 S ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. On the 30 S subunit, for example, the A site (aminoacyl site) close to the 3′-end of 16 S rRNA is highly important in the decoding process. Binding by some aminoglycoside antibiotics to the A site leads to erroneous protein synthesis and is lethal for bacteria. We targeted the A site on purified 30 S ribosomal subunits from Escherichia coli with a set of overlapping, complementary OMe (2′-O-methyl) 10-mer oligoribonucleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleotides. We show that there is a single high-affinity region, spanning from A1493 to C1510 (Kd, 29–130 nM), flanked by two lower-affinity regions, within a span from U1485 to G1516 (Kd, 310–4300 nM). Unexpectedly, addition of the aminoglycoside antibiotic paromomycin (but not hygromycin B) caused a dose-dependent increase of up to 7.5-fold in the binding of the highest affinity 10-mer 1493 to 30 S subunits. Oligonucleotides containing residues complementary to A1492 and/or A1493 showed particularly marked stimulation of binding by paromomycin. The results are consistent with high-resolution structures of antibiotic binding to the A site and with greater accessibility of residues of A1492 and A1493 upon paromomycin binding. 10-mer 1493 binding is thus a probe of the conformational switch to the ‘closed’ conformation triggered by paromomycin that is implicated in the discrimination by 30 S subunits of cognate from non-cognate tRNA and the translational misreading caused by paromomycin. Finally, we show that OMe oligonucleotides targeted to the A site are moderately good inhibitors of in vitro translation and that there is a limited correlation of inhibition activity with binding strength to the A site.

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Background: PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay's potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the "field conditions" to attain a clinically useful level of specificity. Methods: The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination. Results: The assay's specificity was 90.5% in CSF collected into the snap-top tubesup from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study. Conclusions: A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay's sensitivity and its negative predictive value.

BackgroundWhilst mild neonatal hyponatraemia is common and relatively harmless, extreme hyponatraemia of 95 mmol per litre has never been reported in a premature baby and such a level could be associated with immediate as well as long-lasting detrimental effects on health.Case presentationTwenty-four days old baby boy born at 28 weeks gestation (triplet one) unexpectedly became moribund with hypovolaemic shock and was found to have blood sodium of 95 mmol per litre. Diagnostic work up revealed a combination of a urinary tract infection, inadvertently low sodium provision with donor breast milk, and weak renin-angiotensin-aldosterone response. Commencement of treatment with intravenous fluids and extra sodium led to unanticipated diuresis and faster than expected increase of sodium level. Ultimately, treatment resulted in clinical recovery and normalisation of sodium level, which subsequently remained normal with no additional sodium supplementation. Follow up revealed mild spastic diplegia.ConclusionContinuous monitoring and daily medical reviews may not be sensitive enough to recognise development of extreme hyponatraemia. Blood sodium levels should be monitored closely and any abnormalities promptly addressed. Treatment of hypovolaemic hyponatraemia should be centred on fluid resuscitation, anticipation of “paradoxical” diuresis, and blood sodium correction rate of 8 to 10 mmol per litre per day.