Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing.

OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.

An oncogenic role of eIF3e/INT6 in human breast cancer.

Altered expression of the eukaryotic translation initiation factor 3 (eIF3) subunit eIF3e/INT6 has been described in various types of human cancer, but the nature of its involvement in tumorigenesis is not yet clear. Using immunohistochemical analysis of 81 primary breast cancers, we found that high tumor grade correlated significantly with elevated cytoplasmic eIF3e level in epithelial tumor cells. Analysis of protein synthesis after siRNA-mediated knockdown in breast cancer cell lines indicated that eIF3e is not required for bulk translation. Microarray analysis of total and polysomal RNAs nonetheless identified distinct sets of mRNAs regulated either positively or negatively by eIF3e; functional classification of these revealed a marked enrichment of genes involved in cell proliferation, invasion and apoptosis. Validated mRNA targets regulated positively at the translational level by eIF3e included urokinase-type plasminogen activator and apoptotic regulator BCL-XL, whereas synthesis of proteins including the mitotic checkpoint component MAD2L1 was negatively regulated. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. Taken together, our study data suggest that eIF3e has a positive role in breast cancer progression. It regulates the translation, and in some cases abundance, of mRNAs involved in key aspects of cancer cell biology.

Functional genomics of pathogenic bacteria.

Microbial diseases remain the commonest cause of global mortality and morbidity. Automated-DNA sequencing has revolutionized the investigation of pathogenic microbes by making the immense fund of information contained in their genomes available at reasonable cost. The challenge is how this information can be used to increase current understanding of the biology of commensal and virulence behaviour of pathogens with particular emphasis on in vivo function and novel approaches to prevention. One example of the application of whole-genome-sequence information is afforded by investigations of the pathogenic role of Haemophilus influenzae lipopolysaccharide and its candidacy as a vaccine.

A putatively phase variable gene (dca) required for natural competence in Neisseria gonorrhoeae but not Neisseria meningitidis is located within the division cell wall (dcw) gene cluster.

A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci.

Genome sequencing and annotation.

The availability of complete microbial genome sequences enormously facilitates experimental molecular investigations of the respective organisms by providing complete lists of genes, their genetic contexts, and their predicted functions. This can be used in a number of ways to focus studies on bacterial pathogenesis and also vaccine development (1,2). The complete genome sequences from two unrelated strains of Neisseria meningitidis, a derivative of isolate MC58 which originally expressed serogroup B capsule and strain Z2491, which is serogroup A, are now available (3,4). The genome sequences of both these strains were determined using the whole genome shotgun approach (5). In this approach, randomly sheared chromosomal DNA is cloned to make a small insert library (1.5-2.0 kb for MC58, 0.5-0.8 kb and 1.0-1.5 kb for Z2491), then each insert is sequenced from both ends using plasmidspecific primers. For the MC58 genome sequence, a large insert lambda library (8-24 kb) was also used. In the initial sequencing phase, 6-8 times coverage of the estimated size of the genome is generally achieved. The DNA sequences are linked together (assembled) into large contigs (a derivative of the word contiguous). Polymerase chain reaction (PCR) and sequencing of large insert libraries are then used to join the contigs, close gaps, and resolve ambiguities (see ref. 6 for a review).

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58.

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases.

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5'-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17-38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.

Bacterial evolution: bacteria play pass the gene.

DNA transfer between related bacterial species is enhanced by species-specific uptake sequences. These sequences have been used to identify genes that have been transferred from Haemophilus to Neisseria, providing a clear example of interspecific transfer of DNA in the evolution of the pathogenic Neisseria.

Simple sequence repeats in the Helicobacter pylori genome.

We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et aL (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase-variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell-surface-associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1-2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H. pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.

Implications of sequencing bacterial genomes for pathogenesis and vaccine development.

Improvements in homology search methodology and functional predictions are being complemented by the increase in the volume of sequence data with which comparative analyses can be performed. The experimental methods needed for investigation of gene function and expression in a variety of model systems of infection continue to develop. The identification of surface-exposed microbial structures and their conservation in natural populations of pathogenic species offers prospects for developing novel vaccines. A major challenge is the development of efficient screening methods to select the most promising candidates, such as immunisation with DNA.

A randomised placebo controlled trial of loop diuretics in moderate/severe pre-eclampsia, following delivery.

Nineteen patients with pre-eclampsia were randomised to receive 40 mg of frusemide or placebo by mouth daily for 7 days in the first post-partum week. Outcome measures included mean and maximum blood pressure, the need for additional antihypertensive treatment during that period and mean length of hospital stay. There were no statistically significant differences in outcome between the treatment and placebo groups although there was a trend to more rapid lowering of blood pressure following delivery in those receiving frusemide.

Controversies: the mature breech should be delivered by elective cesarean section.

The fetal hazards of vaginal breech delivery have been recognised for centuries. In recent years the increased safety of Cesarean delivery has prompted a steady rise in its use for the delivery of the term breech fetus. In many maternity units more than 90% of breech babies are delivered in this way. In contrast some obstetricians argue that with appropriate selection criteria, safe, selective, vaginal breech delivery is possible. This argument is not supported by population studies, which indicate that perinatal mortality and morbidity rates associated with attempts at vaginal breech delivery are between 1-2%.

A prospective laboratory-based audit of gentamicin use and therapeutic monitoring.

We report a study investigating the proportion of patients in whom therapeutic serum concentrations of gentamicin are achieved in the early phase of treatment and to determine the underlying reasons for sub-optimal therapy. A laboratory based prospective study of 83 courses of gentamicin was performed (excluding patients receiving renal replacement therapy or with bacterial endocarditis) in a London teaching hospital. Of 83 monitored courses 74 had paired levels tested. Initial trough concentrations were > 2 mg/L in nine (12%) and were < 1 mg/L in 51 (69%) indicating levels were seldom in the toxic range. The first monitored peaks were sub-therapeutic in 58 (78%) courses using a cut-off of 5 mg/L and only seven (9%) were greater than 6 mg/L. Of these seven patients, six had troughs levels of greater than 2 mg/L. Of those with initial levels below 5 mg/L, 33 had further serum levels tested, of which 26 (79%) remained below 5 mg/L. Of those for whom dosing information was available 73% received 80 mg t.d.s. and the mean dose for those for which body weight was known was 3.3 mg/kg/day (S.D. = 0.7). Most patients continued to receive 8-hourly dosing with 80 mg of gentamicin, leading to subtherapeutic peak levels in the majority of cases. In those patients with satisfactory peaks, such dosing frequently leads to elevated troughs. This suggests that such dosing practices should be abandoned and replaced with dosing based on body weight and divided into no more than two daily doses.