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1.  Multi-Serotype Pneumococcal Nasopharyngeal Carriage Prevalence in Vaccine Naïve Nepalese Children, Assessed Using Molecular Serotyping 
PLoS ONE  2015;10(2):e0114286.
Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.
doi:10.1371/journal.pone.0114286
PMCID: PMC4313945  PMID: 25643355
2.  A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence 
BMC Microbiology  2014;14(1):210.
Background
The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.
Results
Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.
By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.
Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.
Conclusions
We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0210-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-014-0210-x
PMCID: PMC4243769  PMID: 25163487
Streptococcus pneumoniae; cpsE; Capsule; Nonencapsulated; SNP
3.  Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic 
BMC Genomics  2014;15:270.
Background
Leprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England.
Results
Here we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains.
Conclusions
The genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century.
doi:10.1186/1471-2164-15-270
PMCID: PMC4234520  PMID: 24708363
Mycobacterium leprae; SNP; Leprosy; Genome; Ancient DNA; Middle Ages; Bioarchaeology
4.  Multiple Streptococcus pneumoniae Serotypes in Aural Discharge Samples from Children with Acute Otitis Media with Spontaneous Otorrhea 
Journal of Clinical Microbiology  2013;51(10):3409-3411.
Among 55 children with cultures positive for acute otitis media with spontaneous otorrhea, 28 (51%) had cultures positive for aural Streptococcus pneumoniae, and in 10 of these, two distinct strains were detected, in which 5 had pairs of strains that were both capsule-bearing serotypes. Such cases were more likely to have cultures positive for other otopathogens than those with only one pneumococcus present.
doi:10.1128/JCM.01303-13
PMCID: PMC3811614  PMID: 23885003
5.  Defective Pneumococcal-Specific Th1 Responses in HIV-Infected Adults Precedes a Loss of Control of Pneumococcal Colonization 
We show that pneumococcal colonization rates and composition greatly change during human immunodeficiency virus infection. These changes are associated with fewer Th1 pneumococcal-specific responses but unaltered Th17 responses. Altered pneumococcal colonization and impaired pneumococcal-specific immunity are not rectified by highly active antiretroviral therapy.
Background. African adults infected with human immunodeficiency virus (HIV) have high rates of pneumococcal colonization and invasive disease. Here we have investigated the possibility that HIV disrupts the normal balance of pneumococcal-specific helper T cell (Th) 1/Th17 immunity to colonization, resulting in a more permissive nasopharyngeal niche.
Methods. One hundred thirty-six HIV-infected and -uninfected Malawian adults were enrolled in the study. Changes in rates and composition of nasopharyngeal pneumococcal colonization were analyzed using microarray. The underlying pneumococcal-specific Th1/Th17 responses associated with altered pneumococcal colonization were investigated using flow cytometry.
Results. We find that pneumococcal carriage is only modestly increased in asymptomatic HIV-infected Malawian adults but that colonization rates rise dramatically during symptomatic disease (HIVneg 13%, HIVasy 19%, and HIVsym 38%). These rates remain high in subjects established on antiretroviral therapy (ART): 33% (at 6–12 months) and 52% (at 18 months), with HIV-infected individuals carrying a broader range of invasive and noninvasive serotypes compared with HIV-negative controls. The frequency of multiple serotype carriage (>1 serotype HIVneg 26%, HIVasy 30%, HIVsym 31%, HIVART 31%) is not affected. These changes in colonization are associated with generalized CD4 T-cell depletion, impaired antigen-specific proliferation, and a defect in pneumococcal-specific T-cell interferon-γ but not interleukin 17 production.
Conclusions. These data reveal the persistently poor control of pneumococcal colonization in HIV-infected adults following immune ART-mediated reconstitution, highlighting a potential reservoir for person-to-person spread and vaccine escape. Novel approaches to control colonization either through vaccination or through improvements in the quality of immune reconstitution are required.
doi:10.1093/cid/cis842
PMCID: PMC3526250  PMID: 23024291
Human; T Cells; Mucosa; Streptococcus pneumoniae; HIV
6.  Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression 
BMC Genomics  2013;14:710.
Background
Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of £100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB.
Results
We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression.
Conclusions
We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent’ nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype.
doi:10.1186/1471-2164-14-710
PMCID: PMC3856593  PMID: 24134787
Bovine tuberculosis; Mycobacterium bovis; Microarray; Transcript; SNP; Antisense; Macrophage
7.  Dominant Role of Nucleotide Substitution in the Diversification of Serotype 3 Pneumococci over Decades and during a Single Infection 
PLoS Genetics  2013;9(10):e1003868.
Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype.
Author Summary
Streptococcus pneumoniae (‘the pneumococcus’) is a bacterium commonly found asymptomatically in the human nasopharynx that represents a common cause of diseases such as pneumonia, bacteraemia and meningitis. Some strains have been found to exchange DNA with other bacteria at a high rate. However, serotype 3 pneumococci are unusual both in not exhibiting much genetic variation and causing disease with a comparatively high relative rate of mortality. Here we used whole genome sequencing to characterise 82 serotype 3 pneumococci, finding that the majority of the population accumulate variation very slowly. However, comparing two isolates from a single case of disease revealed a small number of mutations had occurred over a short period of time. These resulted in differences in the activity of several genes, including two encoding a drug efflux pump. The pair of isolates was found to differ in their tolerance of different antimicrobial compounds and their behaviour in a mouse model of disease. However, moving the mutation that caused the change in resistance into a distantly-related pneumococcus failed to fully replicate the other changes in behaviour, which indicates that interpretation of the impact of mutations in different strains of diverse bacterial species will be difficult.
doi:10.1371/journal.pgen.1003868
PMCID: PMC3794909  PMID: 24130509
8.  Nontypeable Pneumococcal Isolates Among Navajo and White Mountain Apache Communities: Are These Really a Cause of Invasive Disease? 
Background. Pneumococci could evade pneumococcal conjugate vaccines (PCV) by modifying, mutating, or deleting vaccine-serotype capsule genes or by downregulating capsule production. We sought to assess whether pneumococci that are nontypeable (NT) by the Quellung reaction truly lack capsule genes or are failing to produce capsule in vitro.
Methods. We applied multilocus sequence typing and a microarray for detection of pneumococcal polysaccharide capsule biosynthesis genes to NT carriage (children aged <5 years; years 1997–2000, 2006–2008) and NT invasive disease (IPD) (all ages; years 1994–2007) isolates from Native American communities.
Results. Twenty-seven of 28 (96.4%) NT IPD isolates had sequence types (STs) typically found among typeable IPD isolates and contained whole or fragments of capsule genes that matched known serotypes; 1 NT-IPD isolate had a profile resembling NT carriage isolates. Forty-nine of 76 (64.5%) NT carriage isolates had STs that typically lack capsule genes and were similar to NT carriage isolates found globally.
Conclusions. This is the first documentation of IPD from an NT strain confirmed to lack all known capsule genes. Most NT IPD isolates have or had the capacity to produce capsule, whereas a majority of NT carriage isolates lack this capacity. We found no evidence of pneumococcal adaptation to PCV7 via downregulation or deletion of vaccine-serotype capsule genes.
doi:10.1093/infdis/jis307
PMCID: PMC3415932  PMID: 22536000
9.  Nontypeable Pneumococcal Isolates Among Navajo and White Mountain Apache Communities: Are These Really a Cause of Invasive Disease? 
Background
Pneumococci could evade pneumococcal conjugate vaccines (PCV) by modifying, mutating, or deleting vaccine-serotype capsule genes or by downregulating capsule production. We sought to assess whether pneumococci that are nontypeable (NT) by the Quellung reaction truly lack capsule genes or are failing to produce capsule in vitro.
Methods
We applied multilocus sequence typing and a microarray for detection of pneumococcal polysaccharide capsule biosynthesis genes to NT carriage (children aged <5 years; years 1997–2000, 2006–2008) and NT invasive disease (IPD) (all ages; years 1994–2007) isolates from Native American communities.
Results
Twenty-seven of 28 (96.4%) NT IPD isolates had sequence types (STs) typically found among typeable IPD isolates and contained whole or fragments of capsule genes that matched known serotypes; 1 NT-IPD isolate had a profile resembling NT carriage isolates. Forty-nine of 76 (64.5%) NT carriage isolates had STs that typically lack capsule genes and were similar to NT carriage isolates found globally.
Conclusions
This is the first documentation of IPD from an NT strain confirmed to lack all known capsule genes. Most NT IPD isolates have or had the capacity to produce capsule, whereas a majority of NT carriage isolates lack this capacity. We found no evidence of pneumococcal adaptation to PCV7 via downregulation or deletion of vaccine-serotype capsule genes.
doi:10.1093/infdis/jis307
PMCID: PMC3415932  PMID: 22536000
10.  Comparative phylogenomics of Streptococcus pneumoniae isolated from invasive disease and nasopharyngeal carriage from West Africans 
BMC Genomics  2012;13:569.
Background
We applied comparative phylogenomics (whole genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) to investigate S. pneumoniae isolates from West Africa, with the aim of providing insights into the pathogenicity and other features related to the biology of the organism. The strains investigated comprised a well defined collection of 58 invasive and carriage isolates that were sequenced typed and included eight different S. pneumoniae serotypes (1, 3, 5, 6A, 11, 14, 19 F and 23 F) of varying invasive disease potential.
Results
The core genome of the isolates was estimated to be 38% and was mainly represented by gene functional categories associated with housekeeping functions. Comparison of the gene content of invasive and carriage isolates identified at least eleven potential genes that may be important in virulence including surface proteins, transport proteins, transcription factors and hypothetical proteins. Thirteen accessory regions (ARs) were also identified and did not show any loci association with the eleven virulence genes. Intraclonal diversity (isolates of the same serotype and MLST but expressing different patterns of ARs) was observed among some clones including ST 1233 (serotype 5), ST 3404 (serotype 5) and ST 3321 (serotype 14). A constructed phylogenetic tree of the isolates showed a high level of heterogeneity consistent with the frequent S. pneumoniae recombination. Despite this, a homogeneous clustering of all the serotype 1 strains was observed.
Conclusions
Comparative phylogenomics of invasive and carriage S. pneumoniae isolates identified a number of putative virulence determinants that may be important in the progression of S. pneumoniae from the carriage phase to invasive disease. Virulence determinants that contribute to S. pneumoniae pathogenicity are likely to be distributed randomly throughout its genome rather than being clustered in dedicated loci or islands. Compared to other S. pneumoniae serotypes, serotype 1 appears most genetically uniform.
doi:10.1186/1471-2164-13-569
PMCID: PMC3534514  PMID: 23107513
11.  Decrease in Pneumococcal Co-Colonization following Vaccination with the Seven-Valent Pneumococcal Conjugate Vaccine 
PLoS ONE  2012;7(1):e30235.
Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.
doi:10.1371/journal.pone.0030235
PMCID: PMC3257259  PMID: 22253924
12.  Stationary phase gene expression of Mycobacterium tuberculosis following a progressive nutrient depletion: a model for persistent organisms?,  
Tuberculosis (Edinburgh, Scotland)  2004;84(3-4):228-238.
Summary
The majority of individuals infected with TB develop a latent infection, in which organisms survive within the body while evading the host immune system. Such persistent bacilli are capable of surviving several months of combinatorial antibiotic treatment. Evidence suggests that stationary phase bacteria adapt to increase their tolerance to environmental stresses. We have developed a unique in vitro model of dormancy based on the characterization of a single, large volume fermenter culture of M. tuberculosis, as it adapts to stationary phase. Cells are maintained in controlled and defined aerobic conditions (50% dissolved oxygen tension), using probes that measure dissolved oxygen tension, temperature, and pH. Microarray analysis has been used in conjunction with viability and nutrient depletion assays to dissect differential gene expression. Following exponential phase growth the gradual depletion of glucose/glycerol resulted in a small population of survivors that were characterized for periods in excess of 100 days. Bacilli adapting to nutrient depletion displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the up-regulation of genes involved in β-oxidation of fatty acids and virulence. A reduced population of non-replicating bacilli went on to adapt sufficiently to re-initiate cellular division.
doi:10.1016/j.tube.2003.12.010
PMCID: PMC3195342  PMID: 15207492
Tuberculosis; Persistence; Metabolism; Microarray; Stationary phase
13.  BμG@Sbase—a microbial gene expression and comparative genomic database 
Nucleic Acids Research  2011;40(Database issue):D605-D609.
The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future.
doi:10.1093/nar/gkr796
PMCID: PMC3245117  PMID: 21948792
14.  The Distribution of Mobile Genetic Elements (MGEs) in MRSA CC398 Is Associated with Both Host and Country 
Genome Biology and Evolution  2011;3:1164-1174.
Methicillin-resistant Staphylococcus aureus clonal complex (CC) 398 has emerged from pigs to cause human infections in Europe and North America. We used a new 62-strain S. aureus microarray (SAM-62) to compare genomes of isolates from three geographical areas (Belgium, Denmark, and Netherlands) to understand how CC398 colonizes different mammalian hosts. The core genomes of 44 pig isolates and 32 isolates from humans did not vary. However, mobile genetic element (MGE) distribution was variable including SCCmec. φ3 bacteriophage and human specificity genes (chp, sak, scn) were found in invasive human but not pig isolates. SaPI5 and putative ruminant specificity gene variants (vwb and scn) were common but not pig specific. Virulence and resistance gene carriage was host associated but country specific. We conclude MGE exchange is frequent in CC398 and greatest among populations in close contact. This feature may help determine epidemiological associations among isolates of the same lineage.
doi:10.1093/gbe/evr092
PMCID: PMC3205603  PMID: 21920902
methicillin-resistant Staphylococcus aureus; host specificity; mobile genetic elements; zoonoses; bacteriophages
15.  Characterization of two in vivo-expressed methyltransferases of the Mycobacterium tuberculosis complex: antigenicity and genetic regulation 
Microbiology (Reading, England)  2008;154(Pt 4):1059-1067.
Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli.
doi:10.1099/mic.0.2007/014548-0
PMCID: PMC3145104  PMID: 18375799
16.  Mycobacterial Cells Have Dual Nickel-Cobalt Sensors 
The Journal of biological chemistry  2007;282(44):32298-32310.
A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was confirmed. KmtR is the second, to NmtR, characterized ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, whereas NmtR retains a response to nickel and cobalt under these conditions. KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases. KmtR has none of the previously defined metal-sensing sites. Substitution of His88, Glu101, His102, His110, or His111 with Gln generated KmtR variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory motifs revealing that only the latter should be used to predict metal sensing.
doi:10.1074/jbc.M703451200
PMCID: PMC3145109  PMID: 17726022
17.  Effect of FliK mutation on the transcriptional activity of the σ54 sigma factor RpoN in Helicobacter pylori 
Microbiology (Reading, England)  2009;155(Pt 6):1901-1911.
Helicobacter pylori is a motile Gram-negative bacterium that colonizes and persists in the human gastric mucosa. The flagellum gene regulatory circuitry of H. pylori is unique in many aspects compared with the Salmonella/Escherichia coli paradigms, and some regulatory checkpoints remain unclear. FliK controls the hook length during flagellar assembly. Microarray analysis of a fliK-null mutant revealed increased transcription of genes under the control of the σ54 sigma factor RpoN. This sigma factor has been shown to be responsible for transcription of the class II flagellar genes, including flgE and flaB. No genes higher in the flagellar hierarchy had altered expression, suggesting specific and localized FliK-dependent feedback on the RpoN regulon. FliK thus appears to be involved in three processes: hook-length control, export substrate specificity and control of RpoN transcriptional activity.
doi:10.1099/mic.0.026062-0
PMCID: PMC3145110  PMID: 19383688
18.  Advanced significance analysis of microarray data based on weighted resampling: a comparative study and application to gene deletions in Mycobacterium bovis 
Bioinformatics (Oxford, England)  2004;20(3):357-363.
Motivation
When analyzing microarray data, non-biological variation introduces uncertainty in the analysis and interpretation. In this paper we focus on the validation of significant differences in gene expression levels, or normalized channel intensity levels with respect to different experimental conditions and with replicated measurements. A myriad of methods have been proposed to study differences in gene expression levels and to assign significance values as a measure of confidence. In this paper we compare several methods, including SAM, regularized t-test, mixture modeling, Wilk’s lambda score and variance stabilization. From this comparison we developed a weighted resampling approach and applied it to gene deletions in Mycobacterium bovis.
Results
We discuss the assumptions, model structure, computational complexity and applicability to microarray data. The results of our study justified the theoretical basis of the weighted resampling approach, which clearly outperforms the others.
Availability
Algorithms were implemented using the statistical programming language R and available on the author’s web-page.
doi:10.1093/bioinformatics/btg417
PMCID: PMC3128991  PMID: 14960462
19.  Global network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus 
BMC Systems Biology  2011;5:68.
Background
Staphylococcus aureus is a major human pathogen and strains resistant to existing treatments continue to emerge. Development of novel treatments is therefore important. Antimicrobial peptides represent a source of potential novel antibiotics to combat resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA). A promising antimicrobial peptide is ranalexin, which has potent activity against Gram-positive bacteria, and particularly S. aureus. Understanding mode of action is a key component of drug discovery and network biology approaches enable a global, integrated view of microbial physiology, including mechanisms of antibiotic killing. We developed a systems-wide functional association network approach to integrate proteome and transcriptome profiles, enabling study of drug resistance and mode of action.
Results
The functional association network was constructed by Bayesian logistic regression, providing a framework for identification of antimicrobial peptide (ranalexin) response modules from S. aureus MRSA-252 transcriptome and proteome profiling. These signatures of ranalexin treatment revealed multiple killing mechanisms, including cell wall activity. Cell wall effects were supported by gene disruption and osmotic fragility experiments. Furthermore, twenty-two novel virulence factors were inferred, while the VraRS two-component system and PhoU-mediated persister formation were implicated in MRSA tolerance to cationic antimicrobial peptides.
Conclusions
This work demonstrates a powerful integrative approach to study drug resistance and mode of action. Our findings are informative to the development of novel therapeutic strategies against Staphylococcus aureus and particularly MRSA.
doi:10.1186/1752-0509-5-68
PMCID: PMC3123200  PMID: 21569391
20.  Improved Detection of Nasopharyngeal Cocolonization by Multiple Pneumococcal Serotypes by Use of Latex Agglutination or Molecular Serotyping by Microarray▿† 
Journal of Clinical Microbiology  2011;49(5):1784-1789.
Identification of Streptococcus pneumoniae in the nasopharynx is critical for an understanding of transmission, estimates of vaccine efficacy, and possible replacement disease. Conventional nasopharyngeal swab (NPS) culture and serotyping (the WHO protocol) is likely to underestimate multiple-serotype carriage. We compared the WHO protocol with methods aimed at improving cocolonization detection. One hundred twenty-five NPSs from an infant pneumococcal-carriage study, containing ≥1 serotype by WHO culture, were recultured in duplicate. A sweep of colonies from one plate culture was serotyped by latex agglutination. DNA extracted from the second plate was analyzed by S. pneumoniae molecular-serotyping microarray. Multiple serotypes were detected in 11.2% of the swabs by WHO culture, 43.2% by sweep serotyping, and 48.8% by microarray. Sweep and microarray were more likely to detect multiple serotypes than WHO culture (P < 0.0001). Cocolonization detection rates were similar between microarray and sweep, but the microarray identified the greatest number of serotypes. A common serogroup type was identified in 95.2% of swabs by all methods. WHO methodology significantly underestimates multiple-serotype carriage compared to these alternate methods. Sweep serotyping is cost-effective and field deployable but may fail to detect serotypes at low abundance, whereas microarray serotyping is more costly and technology dependent but may detect these additional minor carried serotypes.
doi:10.1128/JCM.00157-11
PMCID: PMC3122683  PMID: 21411589
21.  Empirical Bayesian models for analysing molecular serotyping microarrays 
BMC Bioinformatics  2011;12:88.
Background
Microarrays offer great potential as a platform for molecular diagnostics, testing clinical samples for the presence of numerous biomarkers in highly multiplexed assays. In this study applied to infectious diseases, data from a microarray designed for molecular serotyping of Streptococcus pneumoniae was used, identifying the presence of any one of 91 known pneumococcal serotypes from DNA extracts. This microarray incorporated oligonucleotide probes for all known capsular polysaccharide synthesis genes and required a statistical analysis of the microarray intensity data to determine which serotype, or combination of serotypes, were present within a sample based on the combination of genes detected.
Results
We propose an empirical Bayesian model for calculating the probabilities of combinations of serotypes from the microarray data. The model takes into consideration the dependencies between serotypes, induced by genes they have in common, and by homologous genes which, although not identical, are similar to each other in sequence. For serotypes which are very similar in capsular gene composition, extra probes are included on the microarray, providing additional information which is integrated into the Bayesian model. For each serotype combination with high probability, a second model, a Bayesian random effects model is applied to determine the relative abundance of each serotype.
Conclusions
To assess the accuracy of the proposed analysis we applied our methods to experimental data from samples containing individual serotypes and samples containing combinations of serotypes with known levels of abundance. All but two of the known serotypes of S. pneumoniae that were tested as individual samples could be uniquely determined by the Bayesian model. The model also enabled the presence of combinations of serotypes within samples to be determined. Serotypes with very low abundance within a combination of serotypes can be detected (down to 2% abundance in this study). As well as detecting the presence of serotype combinations, an approximate measure of the percentage abundance of the serotypes within the combination can be obtained.
doi:10.1186/1471-2105-12-88
PMCID: PMC3076268  PMID: 21453458
22.  The minor groove-binding agent ELB-21 forms multiple interstrand and intrastrand covalent cross-links with duplex DNA and displays potent bactericidal activity against methicillin-resistant Staphylococcus aureus 
Objectives
The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.
Methods
Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.
Results
ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.
Conclusions
The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.
doi:10.1093/jac/dkr044
PMCID: PMC3073633  PMID: 21393142
antistaphylococcal activity; pyrrolobenzodiazepine dimer; DNA cross-linking; DNA adduct formation; MRSA
23.  Genetic Variation in Spatio-Temporal Confined USA300 Community-Associated MRSA Isolates: A Shift from Clonal Dispersion to Genetic Evolution? 
PLoS ONE  2011;6(2):e16419.
Introduction
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA.
Materials and Methods
Hierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements.
Conclusion
The genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone.
doi:10.1371/journal.pone.0016419
PMCID: PMC3033884  PMID: 21326601
24.  Detection of Large Numbers of Pneumococcal Virulence Genes in Streptococci of the Mitis Group ▿ †  
Journal of Clinical Microbiology  2010;48(8):2762-2769.
Seven streptococcal isolates from the mitis group were analyzed for the presence of pneumococcal gene homologues by comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuraminidase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 mitis group isolates, respectively. The mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), two S. mitis isolates from the nasopharynxes of children, one S. mitis isolate from a case of infective endocarditis, one S. mitis isolate from a dental abscess, and one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynxes of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67 and 82% of pneumococcal virulence genes, S. oralis hybridized to 83% of pneumococcal virulence genes, and S. pseudopneumoniae hybridized to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly), and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the mitis group are closely clustered together, sharing 99.4 to 99.7% sequence identity with pneumococcal nanA alleles.
doi:10.1128/JCM.01746-09
PMCID: PMC2916619  PMID: 20519466
25.  Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans 
Microbiology (Reading, England)  2004;150(Pt 5):1519-1527.
Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notably M. microti shows lower virulence for humans than M. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M. microti strains. Compared to M. tuberculosis H37Rv, 13 deletions were identified in 12 strains of M. microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG. In addition, four new deleted regions, named MiD1, RD1β, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 of M. bovis BCG and M. microti is thought to be crucial for attenuation, in this study, three of the four M. microti strains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M. microti strains. These deletions might therefore have some relation to the different host range of M. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M. microti.
PMCID: PMC2964484  PMID: 15133113

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