Catherine Arnold

Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing

Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database (http://pubmlst.org/neisseria) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis, was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.

Dynamics of lamivudine-resistant hepatitis B virus during adefovir monotherapy versus lamivudine plus adefovir combination therapy.

Adefovir dipivoxil has been used alone or together with lamivudine to suppress lamivudine‐resistant hepatitis B virus (HBV). However, the dynamics of HBV populations under different selection pressures and their impact on treatment outcome are poorly understood. Pyrosequencing® was applied to quantify longitudinally the evolution of wild type and lamivudine/ adefovir‐resistant HBV. Eight patients, with lamivudine‐resistant HBV, were randomized to receive adefovir monotherapy or adefovir/lamivudine combination therapy for a median of 79 and 71 weeks, respectively. Pyrosequencing® proved highly sensitive with a lower limit of quantitation of minor HBV populations of 2% irrespective of viraemia levels. Adefovir/lamivudine treatment resulted in greater viraemia reduction than adefovir monotherapy. During combination therapy, lamivudine‐resistant HBV populations (codons 180 and 204) remained dominant (>90%) and no adefovir‐resistance developed. During adefovir monotherapy, reversion to wild‐type HBV was detected in two patients with one patient accumulating rapidly adefovir‐resistant HBV along with increased viraemia. In conclusion, the dynamics of drug‐resistant HBV strains vary under different selection pressures which have a significant impact on the success of rescue therapy, as well as for the selection of new mutations. The use of techniques such as Pyrosequencing provides an evidence‐based approach for successful management of drug‐resistant HBV. J. Med. Virol. 80: 1160–1170, 2008.

Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping

Background Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. Methods and Findings In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing <8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts ≤ 42 cycles in internal transcribed spacer-real time PCR. Conclusions These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).

Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH

OBJECTIVES The aim of this study was to gain an insight into the molecular mechanisms of the evolution of rifampicin resistance in response to controlled changes in the environment. METHODS We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2. RESULTS The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10(-8) and 8.9 x 10(-8); however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12. CONCLUSIONS The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive.

Real-Time PCRs and Fingerprinting Assays for the Detection and Characterization of Salmonella Genomic Island-1 Encoding Multidrug Resistance: Application to 445 European Isolates of Salmonella, Escherichia coli, Shigella, and Proteus

Salmonella Genomic Island-1 (SGI-1) harbors a cluster of genes encoding multidrug resistance (MDR). SGI-1 is horizontally transmissible and is therefore of significant public health concern. This study presents two novel realtime PCRs detecting three SGI-1 protein-coding genes and a SGI-1 fingerprinting assay. These assays were applied to 445 European enterobacterial isolates. Results from real-time PCRs were comparable to those obtained from gelbased PCRs used for the detection of SGI-1, but were rapid to perform and suitable for large-scale screening. Furthermore, real-time PCRs also detected SGI-1 even when only part of the island was present in bacterial isolates. No trace of SGI-1 was detected in isolates other than Salmonella enterica. The fingerprints showed that regions of SGI-1 outside the MDR region exhibited genomic variations between isolates. In conclusion, the realtime PCRs described here are suitable for the detection of SGI-1 in bacterial isolates. Further studies are necessary to elucidate divergence in its non-MDR region.

Comparison of four-colour IS6110-fAFLP with the classic IS6110-RFLP on the ability to detect recent transmission in the city of Barcelona, Spain

The purpose of the study was to compare the IS6110-RFLP (RFLP) results obtained in a previous epidemiological study in the city of Barcelona, Spain [Borrell S, Espanol M, Orcau A, Tudo G, March F, Cayla JA, et al. Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain. J Clin Microbiol 2009 Jan;47(1):198-204.] with the results obtained with IS6110-fAFLP, [Thorne N, Evans JT, Smith EG, Hawkey PM, Gharbia S, Arnold C. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110-restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clin Microbiol Infect 2007 Oct;13(10):964-70.] on the ability to detect recent transmission. fAFLP was applied to DNA samples of RFLP clustered strains, with and without known epidemiological links, with the additional inclusion of four nucleotide-specific fluorophores to further increase the discrimination of the fragments obtained. Four-colour fAFLP was performed on 123 RFLP clustered strains with no epidemiological link (NELC) and on 28 epidemiologically linked RFLP clustered (ELC) strains grouped into 48 and 13 clusters respectively. Clustering results obtained by the two methods were highly congruent in ELC strains with fAFLP allocating 92.3% of the ELCs. For the NELCs, RFLP results were confirmed in 39/48 (81.2%) of fAFLP-clusters with 0-1 different fragments and 9/48 (18.8%) differed in 2-4 fragments, which are considered genetically related but not recently transmitted. In conclusion, overestimation of recent tuberculosis transmission can occur because of the inaccurate analysis of RFLP results. Four-colour fAFLP allows us to differentiate between recent transmission strains and epidemiologically unrelated but genetically related strains.

Insertion site mapping for repeated elements in Mycobacterium tuberculosis

Insertion elements not only act as genetic markers for differentiation of bacteria but their movement in bacterial genomes likely plays an essential role in changing the physical and biochemical traits of the organisms when adapting to new environments. Genomic Insertion Site mapping of transposable elements could shed light on the putative altered function of adjacent genes. In the era of whole genome sequencing where repeat elements are difficult to sequence with short read technologies and in the absence of high throughput technologies especially in poorer resource settings, an alternative approach to their characterisation is needed. A rapid and simple method of insertion site mapping that uses Insertion Sequence 6110 (IS6110) fluorescent amplified fragment length polymorphism (FAFLP) PCR as a foundation and then uses additional selective bases to reduce the number of fragments generated was developed. This was applied to Mycobacterium tuberculosis H37Rv sequenced strain to compare the experimental data with the in silico results. This was successfully achieved for all but two of the sixteen fragments generated by FAFLP and demonstrated that, by using this technique, insertion sites can be mapped onto the genomes of M. tuberculosis.

Mapping the genetic diversity within major clonal complexes of meticillin-resistant Staphylococcus aureus utilizing genome-wide fluorescent amplified fragment length polymorphism markers

The genetic diversity between major meticillin-resistant Staphylococcus aureus (MRSA) lineages was probed using fluorescent amplified fragment length polymorphism (FAFLP) as a random genome sampling tool. Genomic DNA was digested with endonucleases BglII and Csp6I and a subset of the restricted fragments were amplified using the primer pair BglII+A and Csp6I+0. Sixty-seven FAFLP profiles consisting of 46-68 amplified fragments ranging in size from 50 to 600 bp were exhibited amongst the 71 isolates analysed. Cluster analysis of FAFLP data revealed concordance with spa typing and MLST clonal complexes (CC), with isolates of each CC grouping in the same FAFLP cluster. Furthermore, FAFLP could differentiate subtypes within the homogeneous CC22 isolates and also between MLST sequence types 8 and 239. The discriminatory power of FAFLP was 0.998 compared to values of 0.975 and 0.909 for spa typing and MLST, respectively. Thus, FAFLP analysis proved to be a rapid, reproducible and high-resolution tool that displayed the microheterogeneity within MRSA lineages. Using FAFLP data, lineage-specific fragments were identified and sequenced; these encoded toxins, antibiotic resistance determinants and bacteriophage resistance factors. Lineage-specific sequence variations were observed, which may provide insights into the evolution and fitness of successful lineages. This will also aid in the development of rapid and high-throughput diagnostic PCR-based assays for the identification of MRSA lineages in resource-poor settings.

Rapid Analysis of Resistant Mutant Genotypes Using Pyrosequencing

The rapid detection of nucleotide mutations conferring drug resistance is especially important for organisms with long generation times. Mycobacterium tuberculosis, an organism thought to infect around one-third of the global population, is probably the most important of these slow-growers. Multidrug-resistant (MDR) strains of M. tuberculosis, indicated by resistance to rifampicin, are emerging and their rapid detection is crucial for treatment and control of this pathogen. Single nucleotide polymorphisms (SNPs) in the rifampicin resistance determining region (RRDR) of rpoB, an 81-bp region of the RNA polymerase B gene, confer resistance to rifampicin in the vast majority of cases. Combining PCR of this region and rapid short read sequencing using Pyrosequencing enables rapid high-throughput MDR strains to be detected from sputum and early cultures.

Sequence analysis of the rifampicin resistance determining region (RRDR) of rpoB gene in multidrug resistance confirmed and newly diagnosed tuberculosis patients of Punjab, Pakistan

Molecular screening of new patients suspected for TB could help in the effective control of TB in Pakistan as it is a high TB burden country. It will be informative to understand the prevalence of multi drug resistance for a better drug regimen management in this geographical area. The Rifampicin resistance determining region (RRDR) sequencing was used to identify mutations associated with drug resistance in DNA extracts from 130 known multidrug resistant (MDR) cultured strains and compared with mutations observed in DNA extracts directly from 86 sputum samples from consecutive newly diagnosed cases in Lahore, Pakistan. These newly diagnosed samples were positive for smear microscopy, chest X-ray and presumed sensitive to first line drugs. In the known MDR group the most frequent mutations conferring resistance were found in rpoB531 (n = 51, 39.2%). In the newly diagnosed tuberculosis group with no history of MDR, mutations in rpoB531 were seen in 10 of the samples (11.6%). Collectively, all mutations in the RRDR region studied were observed in 80 (61.5%) of known MDR cases and in 14 (16.3%) of the newly diagnosed cases. Using the RRDR as a surrogate marker for MDR, sequences for the newly diagnosed (presumed sensitive) group indicate much higher levels of MDR than the 3.9% WHO 2015 global estimate and suggests that molecular screening directly from sputum is urgently required to effectively address the detection and treatment gaps to combat MDR in this high burden country.