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1.  Chlamydia trachomatis from Australian Aboriginal people with trachoma are polyphyletic composed of multiple distinctive lineages 
Nature Communications  2016;7:10688.
Chlamydia trachomatis causes sexually transmitted infections and the blinding disease trachoma. Current data on C. trachomatis phylogeny show that there is only a single trachoma-causing clade, which is distinct from the lineages causing urogenital tract (UGT) and lymphogranuloma venerum diseases. Here we report the whole-genome sequences of ocular C. trachomatis isolates obtained from young children with clinical signs of trachoma in a trachoma endemic region of northern Australia. The isolates form two lineages that fall outside the classical trachoma lineage, instead being placed within UGT clades of the C. trachomatis phylogenetic tree. The Australian trachoma isolates appear to be recombinants with UGT C. trachomatis genome backbones, in which loci that encode immunodominant surface proteins (ompA and pmpEFGH) have been replaced by those characteristic of classical ocular isolates. This suggests that ocular tropism and association with trachoma are functionally associated with some sequence variants of ompA and pmpEFGH.
Chlamydia trachomatis isolates causing a blinding disease (trachoma) form a single lineage that is different from the lineages causing urogenital infections. Here, Andersson et al. show however that trachoma isolates from Australia are more closely related to urogenital strains than to other trachoma isolates.
PMCID: PMC4773424  PMID: 26912299
2.  Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis 
PLoS ONE  2016;11(2):e0149011.
The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.
PMCID: PMC4752267  PMID: 26871455
3.  Molecular characterisation of the Chlamydia pecorum plasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution 
PeerJ  2016;4:e1661.
Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (pCpec) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that pCpec is common, but not ubiquitous in C. pecorum from all of the infected hosts.
Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel pCpecs in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala.
Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 pCpecs show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight pCpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 pCpec sequences resolved plasmids into 12 distinct genotypes, with five shared between pCpecs from different isolates, and the remaining seven genotypes being unique to a single pCpec. Phylogenetic analysis revealed congruency and co-evolution of pCpecs with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum. This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate the function of this putative C. pecorum virulence factor.
PMCID: PMC4748734  PMID: 26870613
Chlamydia pecorum; Molecular characterisation; Comparative analyses; Plasmid; Phylogeny; Koala
4.  Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations 
Graphical abstract
Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.
PMCID: PMC4820968  PMID: 26746581
−p, absence of virulence plasmid (either pSS or pINV); +p, presence of virulence plasmid (either pSS or pINV); Cyto, cytoplasm; CytoM, cytoplasmic membrane; DOMV, detergent-derived outer membrane vesicles; E. coli, Escherichia coli; GMMA, Generalized Modules for Membrane Antigens; NOMV, native outer membrane vesicles; OM, outer membrane; OM lipo, outer membrane lipoprotein; OMV, outer membrane vesicles; OAg, O antigen; PP, periplasm; SD, standard deviation; Sf2a, hyperblebbing plasmid-cured OAg-deficient Shigella flexneri 2a 2457T; SHIFL, Shigella flexneri protein database, mixed serotypes; SHIF8, Shigella flexneri serotype 5b protein database; SHISO, Shigella sonnei 53G protein database; SHISS, Shigella sonnei 046 protein database; SOMV, spontaneous outer membrane vesicles; Ss, hyperblebbing plasmid-cured OAg-deficient Shigella sonnei 53G; vs, versus; Shigella sonnei; Shigella flexneri 2a; Proteomics; GMMA; Vaccine; iBAQ
5.  Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp. 
Scientific Reports  2015;5:17609.
Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.
PMCID: PMC4671022  PMID: 26639610
6.  Types of Relational Aggression in Girls Are Differentiated by Callous-Unemotional Traits, Peers and Parental Overcontrol 
Behavioral Sciences  2015;5(4):518-536.
Adolescent girls often perpetrate aggression by gossiping and spreading rumours about others, by attempting to ruin relationships and by manipulating and excluding others. Further, males and females engage in reactive and proactive relational aggression differently. In this study, we examined the individual, peer and parental contextual factors that best explained the use of reactive and proactive relational aggression in girls. Female participants (n = 614; ages 11–18 years) completed questionnaires on aggression, callous-unemotional (CU) traits, delinquency, peer delinquency, gender composition of their peer group, resistance to peer influence and perceived parental overcontrol. Multinomial logistic regression was used to examine the effects of individual, peer- and parent-related variables on the likelihood of being classified as a low aggressor, reactive aggressor or proactive/reactive aggressor. Girls in the combined reactive/proactive aggression group were younger, had greater CU traits, a lower proportion of male peers and greater perception of parental control than both the reactive and low aggressive groups. Both highly aggressive groups were more delinquent and had greater peer delinquency than the low aggressive group. This study suggests those girls who show relational aggression for the purpose of gaining status and revenge feel restrained by their parents and may gravitate toward relationships that support their behaviour.
PMCID: PMC4695776  PMID: 26580659
callous-unemotional traits; aggression subtypes; parent-child relationship; peers; females
7.  Vibrio cholerae Serogroup O139: Isolation from Cholera Patients and Asymptomatic Household Family Members in Bangladesh between 2013 and 2014 
PLoS Neglected Tropical Diseases  2015;9(11):e0004183.
Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere.
Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup.
Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups.
These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.
Author Summary
Vibrio cholerae serogroup O1 is thought to be the sole causative agent for cholera in Bangladesh and most of the high risk developing countries. Whilst historically Vibrio cholerae serogroup O139 has been seen to cause sporadic disease, the overall numbers of reported O139 clinical cases are low, with none reported in Bangladesh since 2005. Here we report four patients suffering from cholera attributed to serogroup O139 V. cholerae. Cases 1 and 2 were symptomatic (isolated strains 1, 2), and cases 3 and 4 were asymptomatic (isolated strains 3, 4). All cases were from urban Dhaka and represented a range of age groups. Cases 2–4 presented with no sign of dehydration whereas case 1 showed some signs of dehydration. Phenotypic and whole genome sequence data indicates that one of the four O139 V. cholerae isolates represents a novel O139 subtype. Since natural infection with V. cholerae O1 or vaccination with currently available licensed cholera vaccines (e.g., Dukoral) provides little protection against O139, we conclude that V. cholerae O139 remains in circulation and is still causing a low incidence of cholera. Therefore, further studies looking at the significance of these isolates towards the total burden of cholera in Bangladesh is warranted, including clinical evaluation, genome sequencing and immunobiochemistry.
PMCID: PMC4642977  PMID: 26562418
8.  Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts 
Infection and Immunity  2015;83(5):1778-1788.
The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.
PMCID: PMC4399070  PMID: 25690095
9.  Arresting HIV: Fostering Partnerships between Sex Workers and Police to Reduce HIV Risk and Promote Professionalization within Policing Institutions: A Realist Review 
PLoS ONE  2015;10(10):e0134900.
In many countries around the world sex work is criminalised and its regulatory control is therefore often in the hands of the police. In addition to the impact of this criminalised legal environment, much literature describes the negative impact that certain police practices can have on the ability of sex workers and the programs that work with sex workers to access essential HIV prevention, treatment, care and support services. This situation has resulted in persistent concentrated HIV epidemics among sex workers in many countries of the world. The need for multi-sector partnerships between police and HIV programs is increasingly recognised in various UN declarations and resolutions yet descriptions of the process or key ingredients required to actually establish and sustain these necessary partnerships between police and sex workers [or the programs that provide essential services to sex workers] are sparse. The paper seeks to establish key considerations and critical processes that are required to foster partnerships that if further investigated and scaled up, could result in an enhanced enabling environment for the provision of essential HIV services for sex workers around the globe. This paper is based on a realist review that investigated isolated examples of partnership formation between law enforcement and HIV programs working with sex workers. This methodology research is designed to work with complex social interventions and is based on the emerging 'realist' approach to evaluation. A realist review methodology was chosen given the paucity of relevant literature in this vein and the authors’ familiarity with the grey literature and relationships with experts who work in this sphere. The review found that political and police leadership, civil society strengthening and police reform in relation to HIV, are critical factors and key ingredients in changing the enabling environment in which sex work takes place to ensure that HIV prevention, individual and public health as well as HIV prevention and the promotion of human rights are the number one priority. Further research into this relationship is needed to provide evidence for effective HIV programming with police.
PMCID: PMC4619424  PMID: 26488904
10.  Facilitating a transition from compulsory detention of people who use drugs towards voluntary community-based drug dependence treatment and support services in Asia 
Evidence indicates that detention of people who use drugs in compulsory centers in the name of treatment is common in Cambodia, China, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Thailand, and Vietnam. The expansion of such practices has been costly, has not generated positive health outcomes, and has not reduced supply or demand for illicit drugs. United Nations agencies have convened several consultations with government and civil society stakeholders in order to facilitate a transition to voluntary evidence- and community-based drug dependence treatment and support services.
In an effort to support such efforts, an informal group of experts proposes a three-step process to initiate and accelerate national-level transitions. Specifically, the working group recommends the establishment of a national multisectoral decision-making committee to oversee the development of national transition plans, drug policy reform to eliminate barriers to community-based drug dependence treatment and support services, and the integration of community-based drug dependence treatment in existing national health and social service systems.
In parallel, the working group recommends that national-level transitions should be guided by overarching principles, including ethics, human rights, meaningful involvement of affected communities, and client safety, as well as good governance, transparency, and accountability. The transition also represents an opportunity to review the roles and responsibilities of various agencies across the public health and public security sectors in order to balance the workload and ensure positive results.
The need to accelerate national-level transitions to voluntary community-based drug dependence treatment and support services is compelling—on economic, medical, sustainable community development, and ethical grounds—as extensively documented in the literature. In this context, the expert working group fully endorses initiation of a transition towards voluntary evidence- and community-based drug dependence treatment and support services across the region, as well as the steady scale-down of compulsory centers for drug users.
Components of voluntary community-based drug dependence treatment and support services are being implemented in Cambodia, China, Indonesia, Malaysia, and Thailand. However, significant technical and financial support will be required to be allocated from national budgets and by international development agencies in order to complete the transition and reduce the reliance on detention of people who use drugs in Asia.
PMCID: PMC4608322  PMID: 26470779
Drug dependence; Compulsory treatment; Community-based treatment; Planning; Drug policy reform; Health systems; Principles; Resource mobilization; People who use drugs
11.  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
12.  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
13.  Genetic characterization of three qnrS1-harbouring multidrug-resistance plasmids and qnrS1-containing transposons circulating in Ho Chi Minh City, Vietnam 
Journal of Medical Microbiology  2015;64(Pt 8):869-878.
Plasmid-mediated quinolone resistance (PMQR) refers to a family of closely related genes that confer decreased susceptibility to fluoroquinolones. PMQR genes are generally associated with integrons and/or plasmids that carry additional antimicrobial resistance genes active against a range of antimicrobials. In Ho Chi Minh City (HCMC), Vietnam, we have previously shown a high frequency of PMQR genes within commensal Enterobacteriaceae. However, there are limited available sequence data detailing the genetic context in which the PMQR genes reside, and a lack of understanding of how these genes spread across the Enterobacteriaceae. Here, we aimed to determine the genetic background facilitating the spread and maintenance of qnrS1, the dominant PMQR gene circulating in HCMC. We sequenced three qnrS1-carrying plasmids in their entirety to understand the genetic context of these qnrS1-embedded plasmids and also the association of qnrS1-mediated quinolone resistance with other antimicrobial resistance phenotypes. Annotation of the three qnrS1-containing plasmids revealed a qnrS1-containing transposon with a closely related structure. We screened 112 qnrS1-positive commensal Enterobacteriaceae isolated in the community and in a hospital in HCMC to detect the common transposon structure. We found the same transposon structure to be present in 71.4 % (45/63) of qnrS1-positive hospital isolates and in 36.7 % (18/49) of qnrS1-positive isolates from the community. The resulting sequence analysis of the qnrS1 environment suggested that qnrS1 genes are widely distributed and are mobilized on elements with a common genetic background. Our data add additional insight into mechanisms that facilitate resistance to multiple antimicrobials in Gram-negative bacteria in Vietnam.
PMCID: PMC4635468  PMID: 26272054
14.  A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli 
Pathogens and Disease  2015;73(7):ftv047.
Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.
The authors report on a new adhesin, that they have named coli surface antigen 8 subtype B (CS8B); to date, there are five variants of CS8 undetectable using current primers.
Graphical Abstract Figure.The authors report on a new adhesin, that they have named coli surface antigen 8 subtype B (CS8B); to date, there are five variants of CS8 undetectable using current primers.
PMCID: PMC4535451  PMID: 26187892
Enterotoxigenic Escherichia coli; type IVb pilins; coli surface antigen 8 subtype B; Kenya; colonization factors
15.  Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels 
The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica.
PMCID: PMC4290955  PMID: 25339391
16.  A Qualitative Exploration of the Effects of Increasing the Minimum Purchase Age of Alcohol in Thailand 
Drug and alcohol review  2012;32(1):100-105.
Introduction and Aims
Although prevalence of alcohol consumption has been relatively stable among Thai youth, concerns over alcohol-related harms affecting youth influenced the passage of new laws in early 2008, which made it illegal to sell alcohol to anyone under the age of 20. This qualitative study explored the effects of the law on the purchasing patterns of underage Thai bar patrons, in order to understand the strategies employed by underage youth to circumvent the law.
Design and Methods
A total of 41 in-depth interviews were conducted with 18–19 year old bar patrons in Chiang Mai, Thailand.
Underage Thai bar patrons frequented shops where enforcement was not strict and purchased alcohol, from familiar shopkeepers in their neighborhoods. Participants suggested that purchasing alcohol was relatively easy as long as shopkeepers were driven by the need to make a profit.
Discussion and Conclusions
To address alcohol-related harms, the control law must be enforced in a meaningful way to deter youth from purchasing alcohol. Otherwise, the law will have minimal effectiveness in reducing the harms associated with alcohol.
PMCID: PMC4484578  PMID: 22762793
alcohol law; Thailand; purchasing
17.  Relationship between Distinct African Cholera Epidemics Revealed via MLVA Haplotyping of 337 Vibrio cholerae Isolates 
PLoS Neglected Tropical Diseases  2015;9(6):e0003817.
Since cholera appeared in Africa during the 1970s, cases have been reported on the continent every year. In Sub-Saharan Africa, cholera outbreaks primarily cluster at certain hotspots including the African Great Lakes Region and West Africa.
Methodology/Principal Findings
In this study, we applied MLVA (Multi-Locus Variable Number Tandem Repeat Analysis) typing of 337 Vibrio cholerae isolates from recent cholera epidemics in the Democratic Republic of the Congo (DRC), Zambia, Guinea and Togo. We aimed to assess the relationship between outbreaks. Applying this method, we identified 89 unique MLVA haplotypes across our isolate collection. MLVA typing revealed the short-term divergence and microevolution of these Vibrio cholerae populations to provide insight into the dynamics of cholera outbreaks in each country. Our analyses also revealed strong geographical clustering. Isolates from the African Great Lakes Region (DRC and Zambia) formed a closely related group, while West African isolates (Togo and Guinea) constituted a separate cluster. At a country-level scale our analyses revealed several distinct MLVA groups, most notably DRC 2011/2012, DRC 2009, Zambia 2012 and Guinea 2012. We also found that certain MLVA types collected in the DRC persisted in the country for several years, occasionally giving rise to expansive epidemics. Finally, we found that the six environmental isolates in our panel were unrelated to the epidemic isolates.
To effectively combat the disease, it is critical to understand the mechanisms of cholera emergence and diffusion in a region-specific manner. Overall, these findings demonstrate the relationship between distinct epidemics in West Africa and the African Great Lakes Region. This study also highlights the importance of monitoring and analyzing Vibrio cholerae isolates.
Author Summary
Cholera is caused by the toxigenic bacterium Vibrio cholerae. Since cholera was imported into the West African country of Guinea in 1970, cases have been reported on the continent every year. In Sub-Saharan Africa, cholera occurs in a heterogeneous manner; outbreaks primarily cluster at certain hotspots including the African Great Lakes Region and West Africa. To gain further insight into the mechanisms by which cholera outbreaks emerge and diffuse, we performed genetic analyses of 337 Vibrio cholera isolates from the Democratic Republic of the Congo (DRC), Zambia, Guinea and Togo. Isolates from both patients and environmental samples were examined. Our findings demonstrate the relationship between distinct epidemics in West Africa and the African Great Lakes Region. For example, certain strains in the DRC have circulated in the region over a period of several years, occasionally giving rise to expansive epidemics. We also found that the six environmental isolates in our panel were unrelated to the epidemic isolates. Such insight into the country- and region-specific dynamics of the disease is critical to implement optimized public health strategies to control and prevent cholera epidemics. This study also highlights the importance of analyzing Vibrio cholerae isolates to complement epidemiological studies.
PMCID: PMC4482140  PMID: 26110870
18.  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
19.  Draft genomes of Shigella strains used by the STOPENTERICS consortium 
Gut Pathogens  2015;7:14.
Despite a significant global burden of disease, there is still no vaccine against shigellosis widely available. One aim of the European Union funded STOPENTERICS consortium is to develop vaccine candidates against Shigella. Given the importance of translational vaccine coverage, here we aimed to characterise the Shigella strains being used by the consortium by whole genome sequencing, and report on the stability of strains cultured in different laboratories or through serial passage.
We sequenced, de novo assembled and annotated 20 Shigella strains being used by the consortium. These comprised 16 different isolates belonging to 7 serotypes, and 4 derivative strains. Derivative strains from common isolates were manipulated in different laboratories or had undergone multiple passages in the same laboratory. Strains were mapped against reference genomes to detect SNP variation and phylogenetic analysis was performed.
The genomes assembled into similar total lengths (range 4.14–4.83 Mbp) and had similar numbers of predicted coding sequences (average of 4,400). Mapping analysis showed the genetic stability of strains through serial passages and culturing in different laboratories, as well as varying levels of similarity to published reference genomes. Phylogenetic analysis revealed the presence of three main clades among the strains and published references, one containing the Shigella flexneri serotype 6 strains, a second containing the remaining S. flexneri serotypes and a third comprised of Shigella sonnei strains.
This work increases the number of the publically available Shigella genomes available and specifically provides information on strains being used for vaccine development by STOPENTERICS. It also provides information on the variability among strains maintained in different laboratories and through serial passage. This work will guide the selection of strains for further vaccine development.
PMCID: PMC4454270  PMID: 26042182
Shigella; STOPENTERICS; Genome; Vaccine
20.  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
21.  Antimicrobial Drug Resistance of Vibrio cholerae, Democratic Republic of the Congo 
Emerging Infectious Diseases  2015;21(5):847-851.
We analyzed 1,093 Vibrio cholerae isolates from the Democratic Republic of the Congo during 1997–2012 and found increasing antimicrobial drug resistance over time. Our study also demonstrated that the 2011–2012 epidemic was caused by an El Tor variant clonal complex with a single antimicrobial drug susceptibility profile.
PMCID: PMC4412219  PMID: 25897570
antimicrobial resistance; bacteria; cholera; Vibrio cholerae O1; MLVA; whole-genome sequencing; El Tor variant; clone; Democratic Republic of the Congo
22.  A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage 
Environmental Microbiology  2014;17(4):1090-1102.
Lateral gene transfer (LGT) has been crucial in the evolution of the cholera pathogen, Vibrio cholerae. The two major virulence factors are present on two different mobile genetic elements, a bacteriophage containing the cholera toxin genes and a genomic island (GI) containing the intestinal adhesin genes. Non-toxigenic V. cholerae in the aquatic environment are a major source of novel DNA that allows the pathogen to morph via LGT. In this study, we report a novel GI from a non-toxigenic V. cholerae strain containing multiple genes involved in DNA repair including the recombination repair gene recA that is 23% divergent from the indigenous recA and genes involved in the translesion synthesis pathway. This is the first report of a GI containing the critical gene recA and the first report of a GI that targets insertion into a specific site within recA. We show that possession of the island in Escherichia coli is protective against DNA damage induced by UV-irradiation and DNA targeting antibiotics. This study highlights the importance of genetic elements such as GIs in the evolution of V. cholerae and emphasizes the importance of environmental strains as a source of novel DNA that can influence the pathogenicity of toxigenic strains.
PMCID: PMC4405046  PMID: 24889424
23.  An O Antigen Capsule Modulates Bacterial Pathogenesis in Shigella sonnei 
PLoS Pathogens  2015;11(3):e1004749.
Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.
Author Summary
Shigellosis is a major global health concern. Recently, a shift in the dominance of types of Shigella that cause disease has been observed with S. sonnei increasing in prevalence under improved socio-economic conditions leading to a replacement of S. flexneri. Most of the knowledge of Shigella disease mechanisms has been obtained from studies of S. flexneri. We found that S. sonnei possesses a high molecular weight sugar capsule that is absent in S. flexneri 2a. Removal of the capsule made S. sonnei bacteria highly invasive in vitro and strongly inflammatory in vivo, but in contrast, there was reduced spreading of these mutant bacteria from the gut to peripheral organs in rabbits and higher sensitivity to complement-mediated lysis. Thus, the capsule plays a role in both, invasion and protection of the bacteria against the innate immune defense of the host during the infection. These findings indicate that the capsule is an important virulence factor for S. sonnei. Based on the substantial changes in pathogenesis observed upon removal and overexpression of the capsule, we hypothesize that its level of expression may be under significant evolutionary pressure.
PMCID: PMC4368438  PMID: 25794007
24.  Novel R Pipeline for Analyzing Biolog Phenotypic Microarray Data 
PLoS ONE  2015;10(3):e0118392.
Data produced by Biolog Phenotype MicroArrays are longitudinal measurements of cells’ respiration on distinct substrates. We introduce a three-step pipeline to analyze phenotypic microarray data with novel procedures for grouping, normalization and effect identification. Grouping and normalization are standard problems in the analysis of phenotype microarrays defined as categorizing bacterial responses into active and non-active, and removing systematic errors from the experimental data, respectively. We expand existing solutions by introducing an important assumption that active and non-active bacteria manifest completely different metabolism and thus should be treated separately. Effect identification, in turn, provides new insights into detecting differing respiration patterns between experimental conditions, e.g. between different combinations of strains and temperatures, as not only the main effects but also their interactions can be evaluated. In the effect identification, the multilevel data are effectively processed by a hierarchical model in the Bayesian framework. The pipeline is tested on a data set of 12 phenotypic plates with bacterium Yersinia enterocolitica. Our pipeline is implemented in R language on the top of opm R package and is freely available for research purposes.
PMCID: PMC4365023  PMID: 25786143
25.  A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae 
EMBO Molecular Medicine  2015;7(3):227-239.
Multidrug-resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings.
PMCID: PMC4364942  PMID: 25712531
antimicrobial resistance; bloodstream infections; carbapenemases; Klebsiella pneumoniae; nosocomial infections

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