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1.  The Murray collection of pre-antibiotic era Enterobacteriacae: a unique research resource 
Genome Medicine  2015;7:97.
Studies of historical isolates inform on the evolution and emergence of important pathogens and phenotypes, including antimicrobial resistance. Crucial to studying antimicrobial resistance are isolates that predate the widespread clinical use of antimicrobials. The Murray collection of several hundred bacterial strains of pre-antibiotic era Enterobacteriaceae is an invaluable resource of historical strains from important pathogen groups. Studies performed on the Collection to date merely exemplify its potential, which will only be realised through the continued effort of many scientific groups. To enable that aim, we announce the public availability of the Murray collection through the National Collection of Type Cultures, and present associated metadata with whole genome sequence data for over half of the strains. Using this information we verify the metadata for the collection with regard to subgroup designations, equivalence groupings and plasmid content. We also present genomic analyses of population structure and determinants of mobilisable antimicrobial resistance to aid strain selection in future studies. This represents an invaluable public resource for the study of these important pathogen groups and the emergence and evolution of antimicrobial resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0222-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4584482  PMID: 26411565
2.  Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis 
Nature  2014;514(7523):494-497.
Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact1. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World2. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch3, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.
PMCID: PMC4550673  PMID: 25141181
3.  Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline 
Nature communications  2014;5:4544.
Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.
PMCID: PMC4538795  PMID: 25088811
4.  Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri 
eLife  null;4:e07335.
Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence.
eLife digest
Dysentery is a disease in which the intestine becomes inflamed due to infection by bacteria, viruses or other microbes. Of the bacteria that can cause dysentery, bacteria called Shigella are most often responsible. Humans can acquire Shigella through contaminated food or water. Over the last century, improvements to sanitation combined with access to clean drinking water and better food hygiene have decreased the number of cases of dysentery in many countries. However, the disease continues to be common in low-income countries, especially in young children.
One species of Shigella bacteria, called S. flexneri, causes far more cases of dysentry than other species of Shigella. Across the world, there are many different strains of S. flexneri, but it is not clear how these strains are related to each other, or how variable the genes that they carry are—known as genetic diversity.
Here, Connor, Barker, Baker et al. used a technique called whole genome sequencing to map the evolutionary relationships of over 300 S. flexneri samples collected from around the globe over the past 100 years. This revealed that the bacterial strains can be split into seven groups that each have distinct geographic ranges and combinations of genes that enable the bacteria to infect humans. Many of the strains of bacteria within these groups seem to have colonized, and remained in, quite small geographic areas over long periods of time. This is different to other Shigella species, which appear to have spread between continents far more frequently over much shorter timescales.
Connor, Barker, Baker et al.'s findings reveal that S. flexneri is more diverse than other Shigella bacteria, and suggest that the ability of strains to persist in local areas may have contributed to the species' long-term success. These results point towards the importance of the provision of clean water in the fight against S. flexneri, and underline the need for a greater understanding of how disease-causing bacteria colonize and interact with the local environment.
PMCID: PMC4522646  PMID: 26238191
Shigella; dysentery; genomics; pathogen evolution; E. coli; other
5.  Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation 
Gastroenterology  2015;149(2):456-467.e15.
Background & Aims
Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation.
ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota.
IL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner.
IL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.
PMCID: PMC4539618  PMID: 25917784
UC; CD; Innate Immunity; Immune Regulation; CD, Crohn’s disease; cLPMC, colonic lamina propria mononuclear cell; ELISA, enzyme-linked immunosorbent assay; IBD, inflammatory bowel disease; IL, interleukin; ILC, innate lymphoid cell; IL7R+, IL7R-receptor–positive; mLN, mesenteric lymph node; NCR, natural cytotoxicity receptor; OTU, operational taxonomic unit; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; sIL6Rα, soluble interleukin 6Rα; Th, T-helper cell; TRUC, Tbx21-/-Rag2-/- ulcerative colitis; UC, ulcerative colitis
6.  Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes 
Journal of Antimicrobial Chemotherapy  2015;70(10):2775-2778.
Genome sequencing will be increasingly used in the clinical setting to tailor antimicrobial prescribing and inform infection control outbreaks. A recent technological innovation that could reduce the delay between pathogen sampling and data generation is single molecule sequencing. An example of this technology, which is undergoing evaluation through an early access programme, is the Oxford Nanopore MinION.
We undertook a feasibility study on six clinically significant pathogens, comparing the MinION to the Illumina MiSeq and PacBio RSII platforms. Genomic DNA was prepared and sequenced using the MinION as instructed by the manufacturer, and Illumina MiSeq and PacBio sequencing was performed using established methods.
An evaluation of the accuracy of the MinION based on sequencing of an MRSA isolate showed that error rates were higher in the MinION reads, but provided an even coverage across the entire genome length. The MinION detected all of the expected carbapenemases and ESBL genes in five Gram-negative isolates and the mecA gene in an MRSA isolate.
The MinION can detect the presence of acquired resistance genes, but improvements in accuracy are needed so that antimicrobial resistance associated with mutations in chromosomal genes can be identified.
PMCID: PMC4566964  PMID: 26221019
7.  Whole Genome Sequencing Shows a Low Proportion of Tuberculosis Disease Is Attributable to Known Close Contacts in Rural Malawi 
PLoS ONE  2015;10(7):e0132840.
The proportion of tuberculosis attributable to transmission from close contacts is not well known. Comparison of the genome of strains from index patients and prior contacts allows transmission to be confirmed or excluded.
In Karonga District, Malawi, all tuberculosis patients are asked about prior contact with others with tuberculosis. All available strains from culture-positive patients were sequenced. Up to 10 single nucleotide polymorphisms between index patients and their prior contacts were allowed for confirmation, and ≥ 100 for exclusion. The population attributable fraction was estimated from the proportion of confirmed transmissions and the proportion of patients with contacts.
From 1997–2010 there were 1907 new culture-confirmed tuberculosis patients, of whom 32% reported at least one family contact and an additional 11% had at least one other contact; 60% of contacts had smear-positive disease. Among case-contact pairs with sequences available, transmission was confirmed from 38% (62/163) smear-positive prior contacts and 0/17 smear-negative prior contacts. Confirmed transmission was more common in those related to the prior contact (42.4%, 56/132) than in non-relatives (19.4%, 6/31, p = 0.02), and in those with more intense contact, to younger index cases, and in more recent years. The proportion of tuberculosis attributable to known contacts was estimated to be 9.4% overall.
In this population known contacts only explained a small proportion of tuberculosis cases. Even those with a prior family contact with smear positive tuberculosis were more likely to have acquired their infection elsewhere.
PMCID: PMC4504505  PMID: 26181760
9.  Identification of antigen-specific B cell receptor sequences using public repertoire analysis 
High-throughput sequencing allows detailed study of the B cell receptor (BCR) repertoire post-immunization but it remains unclear to what extent the de novo identification of antigen-specific sequences from the total BCR repertoire is possible. A Hib-MenC-TT conjugate vaccine containing H. influenzae type b (Hib) and group C meningococcal (MenC) polysaccharides as well as tetanus toxoid (TT) was used to investigate the BCR repertoire of adult humans following immunization and test the hypothesis that public or convergent repertoire analysis could identify antigen specific sequences. A number of antigen-specific BCR sequences have previously been reported for Hib and TT which made a vaccine containing these 2 antigens an ideal immunological stimulus. Analysis of identical complementarity determining region (CDR)3 amino acid (AA) sequences that were shared by individuals in the post-vaccine repertoire identified a number of known Hib-specific sequences but only one previously described TT sequence. The extension of this analysis to non-identical but highly similar CDR3 AA sequences revealed a number of other TT-related sequences. The anti-Hib avidity index post-vaccination was strongly correlated with the relative frequency of Hib-specific sequences, indicating that the post-vaccination public BCR repertoire may be related to more conventional measures of immunogenicity correlating with disease protection. Analysis of public BCR repertoire provided evidence of convergent BCR evolution in individuals exposed to the same antigens. If this finding is confirmed, the public repertoire could be used for rapid and direct identification of protective antigen-specific BCR sequences from peripheral blood.
PMCID: PMC4272858  PMID: 25392534
10.  Draft Genome Sequence of the Streptococcus pneumoniae Avery Strain A66 
Genome Announcements  2015;3(3):e00697-15.
We have used HiSeq 2000 technology to generate a draft genome sequence of Streptococcus pneumoniae strain A66. This is a common study strain used in investigations of pneumococcal bacterium-host interactions and was used in the seminal genetic studies of Avery et al.
PMCID: PMC4481291  PMID: 26112793
11.  16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice 
Microbiome  2015;3:26.
Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.
The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1–V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised “universal” PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers.
This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-015-0087-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4482049  PMID: 26120470
16S rRNA gene sequencing; Bifidobacteria; Infant; Intestinal microbiota; FISH
14.  Complete Genome Sequence of Bordetella pertussis D420 
Genome Announcements  2015;3(3):e00657-15.
Bordetella pertussis is the causative agent of whooping cough, a highly contagious, acute respiratory illness that has seen resurgence despite the use of vaccines. We present the complete genome sequence of a clinical strain of B. pertussis, D420, which is representative of a currently circulating clade of this pathogen.
PMCID: PMC4463544  PMID: 26067980
15.  Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis 
BMC Genomics  2015;16(1):447.
Aliivibrio wodanis and Moritella viscosa have often been isolated concurrently from fish with winter-ulcer disease. Little is known about the interaction between the two bacterial species and how the presence of one bacterial species affects the behaviour of the other.
The impact on bacterial growth in co-culture was investigated in vitro, and the presence of A. wodanis has an inhibitorial effect on M. viscosa. Further, we have sequenced the complete genomes of these two marine Gram-negative species, and have performed transcriptome analysis of the bacterial gene expression levels from in vivo samples. Using bacterial implants in the fish abdomen, we demonstrate that the presence of A. wodanis is altering the gene expression levels of M. viscosa compared to when the bacteria are implanted separately.
From expression profiling of the transcriptomes, it is evident that the presence of A. wodanis is altering the global gene expression of M. viscosa. Co-cultivation studies showed that A. wodanis is impeding the growth of M. viscosa, and that the inhibitorial effect is not contact-dependent.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1669-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4462113  PMID: 26059548
Aliivibrio; Moritella; Complete genome; RNA sequencing; Co-culture; Co-infection; Bacteriocin; Winter-ulcer
16.  Locally Confined Clonal Complexes of Mycobacterium ulcerans in Two Buruli Ulcer Endemic Regions of Cameroon 
PLoS Neglected Tropical Diseases  2015;9(6):e0003802.
Mycobacterium ulcerans is the causative agent of the necrotizing skin disease Buruli ulcer (BU), which has been reported from over 30 countries worldwide. The majority of notified patients come from West African countries, such as Côte d’Ivoire, Ghana, Benin and Cameroon. All clinical isolates of M. ulcerans from these countries are closely related and their genomes differ only in a limited number of single nucleotide polymorphisms (SNPs).
Methodology/Principal Findings
We performed a molecular epidemiological study with clinical isolates from patients from two distinct BU endemic regions of Cameroon, the Nyong and the Mapé river basins. Whole genome sequencing of the M. ulcerans strains from these two BU endemic areas revealed the presence of two phylogenetically distinct clonal complexes. The strains from the Nyong river basin were genetically more diverse and less closely related to the M. ulcerans strain circulating in Ghana and Benin than the strains causing BU in the Mapé river basin.
Our comparative genomic analysis revealed that M. ulcerans clones diversify locally by the accumulation of SNPs. Case isolates coming from more recently emerging BU endemic areas, such as the Mapé river basin, may be less diverse than populations from longer standing disease foci, such as the Nyong river basin. Exchange of strains between distinct endemic areas seems to be rare and local clonal complexes can be easily distinguished by whole genome sequencing.
Author Summary
Buruli ulcer (BU) is a progressively necrotizing disease of the skin, caused by infection with Mycobacterium ulcerans. BU occurs very focally with highest incidence in West Africa. The mode of transmission and the nature and role of potential environmental reservoirs are currently not entirely understood. In this study we sequenced whole genomes of sets of M. ulcerans case isolates from two BU endemic regions in Cameroon. We identified two distinct phylogenetic lineages, which directly correlated with the two endemic regions. Furthermore, we showed that the genetic diversity of M. ulcerans is higher in the older endemic region of Cameroon (Nyong river basin) compared to the more recently emerged infection focus in the same country (Mapé river basin). Together, our results demonstrate that M. ulcerans is developing local clonal complexes by the accumulation of single nucleotide polymorphisms (SNPs) and that these complexes often remain confined to individual endemic foci. The gene encoding for rpoB, which is known to harbour drug resistance mutations against rifampicin in M. tuberculosis, was not affected by SNPs in any of the analysed M. ulcerans strains.
PMCID: PMC4457821  PMID: 26046531
17.  Draft Genome Sequence of 24570, the Type Strain of Shigella flexneri 
Genome Announcements  2015;3(3):e00393-15.
Shigella flexneri is a diarrheal pathogen that causes a large disease burden worldwide. We sequenced the genome of the publicly available type strain (S. flexneri 2a strain 24570) of this bacterial species to increase its utility as a reference. We present genome assembly results and comparisons with other reference strains.
PMCID: PMC4447900  PMID: 26021915
18.  Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus 
BMC Genomics  2015;16(1):388.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.
Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.
The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4432960  PMID: 25981586
Staphylococcus aureus; MRSA; ST772; Antibiotic resistance; Mobile genetic elements; Complete genome; DAR4145; India
19.  Mycobacterium abscessus Complex Identification with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry 
Journal of Clinical Microbiology  2015;53(7):2355-2358.
We determined that the Vitek MS Plus matrix-assisted laser desorption ionization–time of flight mass spectrometry using research-use-only (RUO) v.4.12 and in vitro-diagnostic (IVD) v.3.0 databases accurately identified 41 Mycobacterium abscessus subsp. abscessus and 13 M. abscessus subsp. massiliense isolates identified by whole-genome sequencing to the species but not the subspecies level, from Middlebrook 7H11 and Burkholderia cepacia selective agars. Peak analysis revealed three peaks potentially able to differentiate between subspecies.
PMCID: PMC4473197  PMID: 25948607
20.  Emergent and evolving antimicrobial resistance cassettes in community-associated fusidic acid and meticillin-resistant Staphylococcus aureus 
•Resistance to fusidic acid rose among meticillin-resistant Staphylococcus aureus (MRSA) during the 2000s.•Combined surveillance data and whole-genome sequencing analysis dissected changes in the epidemiology of MRSA.•Fusidic acid resistance emerged fastest in the community, where it was used the most.•Resistance occurred in diverse MRSA strains, encoded on staphylococcal cassette chromosome (SCC) elements.•Compact chimeric dual-resistance cassettes indicate a new mechanism for resistance accrual in MRSA.
Fusidic acid is a topical and systemic antimicrobial used for the treatment of staphylococcal infections in hospitals and the community. Sales of fusidic acid and resistance rates among meticillin-resistant Staphylococcus aureus (MRSA) doubled between 1990 and 2001. For the following decade, fusidic acid resistance rates among isolates from Addenbrooke's Hospital (Cambridge, UK) were compared with national resistance rates from MRSA bacteraemia surveillance data and with antimicrobial sales data. Sales of fusidic acid remained relatively constant between 2002 and 2012, whilst fusidic acid resistance increased two- and four-fold in MRSA bacteraemias nationally and in MRSA isolates from Cambridge, respectively. A subgroup of MRSA resistant only to fusidic acid increased after 2006 by 5-fold amongst bacteraemias nationally and 17-fold (to 7.7% in 2012) amongst Cambridge MRSA isolates. All of the available local isolates from 2011 to 2012 (n = 23) were acquired in the community, were not related epidemiologically and belonged to multilocus sequence typing (MLST) groups ST1, 5, 8, 45 or 149 as revealed from analysis of whole-genome sequence data. All harboured the fusC gene on one of six distinct staphylococcal cassette chromosome (SCC) elements, four of which were dual-resistance chimeras that encoded β-lactam and fusidic acid resistance. In summary, fusidic acid-resistant MRSA increased in prevalence during the 2000s with notable rises after 2006. The development of chimeric cassettes that confer dual resistance to β-lactams and fusidic acid demonstrates that the genetics underpinning resistance in community-associated MRSA are evolving.
PMCID: PMC4415905  PMID: 25769787
CA-MRSA; Antimicrobial resistance; Surveillance; SCC genomics
21.  Rapid Emergence of Multidrug Resistant, H58-Lineage Salmonella Typhi in Blantyre, Malawi 
PLoS Neglected Tropical Diseases  2015;9(4):e0003748.
Between 1998 and 2010, S. Typhi was an uncommon cause of bloodstream infection (BSI) in Blantyre, Malawi and it was usually susceptible to first-line antimicrobial therapy. In 2011 an increase in a multidrug resistant (MDR) strain was detected through routine bacteriological surveillance conducted at Queen Elizabeth Central Hospital (QECH).
Longitudinal trends in culture-confirmed Typhoid admissions at QECH were described between 1998–2014. A retrospective review of patient cases notes was conducted, focusing on clinical presentation, prevalence of HIV and case-fatality. Isolates of S. Typhi were sequenced and the phylogeny of Typhoid in Blantyre was reconstructed and placed in a global context.
Between 1998–2010, there were a mean of 14 microbiological diagnoses of Typhoid/year at QECH, of which 6.8% were MDR. This increased to 67 in 2011 and 782 in 2014 at which time 97% were MDR. The disease predominantly affected children and young adults (median age 11 [IQR 6-21] in 2014). The prevalence of HIV in adult patients was 16.7% [8/48], similar to that of the general population (17.8%). Overall, the case fatality rate was 2.5% (3/94). Complications included anaemia, myocarditis, pneumonia and intestinal perforation. 112 isolates were sequenced and the phylogeny demonstrated the introduction and clonal expansion of the H58 lineage of S. Typhi.
Since 2011, there has been a rapid increase in the incidence of multidrug resistant, H58-lineage Typhoid in Blantyre. This is one of a number of reports of the re-emergence of Typhoid in Southern and Eastern Africa. There is an urgent need to understand the reservoirs and transmission of disease and how to arrest this regional increase.
Author Summary
Typhoid fever is a major cause of disease and death around the world, particularly in resource limited settings, although reports suggest that until recently it has been much less prominent in sub-Saharan Africa (SSA) than Asia. Estimates of the precise burden of this disease are, however, difficult, as diagnosis requires advanced laboratory diagnostics. This is a particular problem in much of SSA where long-term laboratory surveillance has been available in just a few settings. Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi is one such setting; between 1998 and 2010, cases of Typhoid fever at QECH were both uncommon and responsive to all antibiotics. In 2011 a marked increase in highly antibiotic resistant Typhoid fever began, with 843 confirmed cases in 2013. A review of cases revealed that one in 40 patients died and one in five had complicated disease. A further study of the DNA of bacteria associated with the outbreak revealed a novel strain, common to Asia, has arrived in Malawi. This is one of a number of reports of the re-emergence of Typhoid fever in Southern and Eastern Africa. There is an urgent need to understand the reservoirs and transmission of disease and how to arrest this regional increase.
PMCID: PMC4409211  PMID: 25909750
22.  Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system 
Genome Biology  2015;16(1):81.
In the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.
We investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.
Our results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4407387  PMID: 25903077
23.  Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis 
Nature Communications  2015;6:6740.
Streptococcus suis causes disease in pigs worldwide and is increasingly implicated in zoonotic disease in East and South-East Asia. To understand the genetic basis of disease in S. suis, we study the genomes of 375 isolates with detailed clinical phenotypes from pigs and humans from the United Kingdom and Vietnam. Here, we show that isolates associated with disease contain substantially fewer genes than non-clinical isolates, but are more likely to encode virulence factors. Human disease isolates are limited to a single-virulent population, originating in the 1920, s when pig production was intensified, but no consistent genomic differences between pig and human isolates are observed. There is little geographical clustering of different S. suis subpopulations, and the bacterium undergoes high rates of recombination, implying that an increase in virulence anywhere in the world could have a global impact over a short timescale.
The bacterium Streptococcus suis causes respiratory tract infections in pigs and meningitis in humans. Here, the authors show that human disease isolates are limited to a single virulent population and find no consistent genomic differences between pig and human isolates.
PMCID: PMC4389249  PMID: 25824154
24.  Capturing the cloud of diversity reveals complexity and heterogeneity of MRSA carriage, infection and transmission 
Nature Communications  2015;6:6560.
Genome sequencing is revolutionizing clinical microbiology and our understanding of infectious diseases. Previous studies have largely relied on the sequencing of a single isolate from each individual. However, it is not clear what degree of bacterial diversity exists within, and is transmitted between individuals. Understanding this ‘cloud of diversity’ is key to accurate identification of transmission pathways. Here, we report the deep sequencing of methicillin-resistant Staphylococcus aureus among staff and animal patients involved in a transmission network at a veterinary hospital. We demonstrate considerable within-host diversity and that within-host diversity may rise and fall over time. Isolates from invasive disease contained multiple mutations in the same genes, including inactivation of a global regulator of virulence and changes in phage copy number. This study highlights the need for sequencing of multiple isolates from individuals to gain an accurate picture of transmission networks and to further understand the basis of pathogenesis.
Populations of bacterial pathogens can be diverse within colonized individuals. Here, the authors sequence the genomes of methicillin-resistant Staphylococcus aureus isolated from staff and animal patients at a veterinary hospital and show considerable within-host diversity that can rise and fall over time.
PMCID: PMC4389252  PMID: 25814293
25.  Selective and Genetic Constraints on Pneumococcal Serotype Switching 
PLoS Genetics  2015;11(3):e1005095.
Streptococcus pneumoniae isolates typically express one of over 90 immunologically distinguishable polysaccharide capsules (serotypes), which can be classified into “serogroups” based on cross-reactivity with certain antibodies. Pneumococci can alter their serotype through recombinations affecting the capsule polysaccharide synthesis (cps) locus. Twenty such “serotype switching” events were fully characterised using a collection of 616 whole genome sequences from systematic surveys of pneumococcal carriage. Eleven of these were within-serogroup switches, representing a highly significant (p < 0.0001) enrichment based on the observed serotype distribution. Whereas the recombinations resulting in between-serogroup switches all spanned the entire cps locus, some of those that caused within-serogroup switches did not. However, higher rates of within-serogroup switching could not be fully explained by either more frequent, shorter recombinations, nor by genetic linkage to genes involved in β–lactam resistance. This suggested the observed pattern was a consequence of selection for preserving serogroup. Phenotyping of strains constructed to express different serotypes in common genetic backgrounds was used to test whether genotypes were physiologically adapted to particular serogroups. These data were consistent with epistatic interactions between the cps locus and the rest of the genome that were specific to serotype, but not serogroup, meaning they were unlikely to account for the observed distribution of capsule types. Exclusion of these genetic and physiological hypotheses suggested future work should focus on alternative mechanisms, such as host immunity spanning multiple serotypes within the same serogroup, which might explain the observed pattern.
Author Summary
Streptococcus pneumoniae is a major respiratory pathogen responsible for a high burden of morbidity and mortality worldwide. Current anti-pneumococcal vaccines target the bacterium’s polysaccharide capsule, of which at least 95 different variants (‘serotypes’) are known, which are classified into ‘serogroups’. Bacteria can change their serotype through genetic recombination, termed ‘switching’, which can allow strains to evade vaccine-induced immunity. By combining epidemiological data with whole genome sequencing, this work finds a robust and unexpected pattern of serotype switching in a sample of bacteria collected following the introduction of routine anti-pneumococcal vaccination: switching was much more likely to exchange one serotype for another within the same serogroup than expected by chance. Several hypotheses are presented and tested to explain this pattern, including limitations of genetic recombination, interactions between the genes that determine serotype and the rest of the genome, and the constraints imposed by bacterial metabolism. This provides novel information on the evolution of S. pneumoniae, particularly regarding how the bacterium might diversify as newer vaccines are introduced.
PMCID: PMC4380333  PMID: 25826208

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