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.
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.
alcohol law; Thailand; purchasing
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.
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.
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.
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.
Aliivibrio; Moritella; Complete genome; RNA sequencing; Co-culture; Co-infection; Bacteriocin; Winter-ulcer
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.
Shigella; STOPENTERICS; Genome; Vaccine
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.
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.
antimicrobial resistance; bacteria; cholera; Vibrio cholerae O1; MLVA; whole-genome sequencing; El Tor variant; clone; Democratic Republic of the Congo
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.
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.
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.
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.
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.
antimicrobial resistance; bloodstream infections; carbapenemases; Klebsiella pneumoniae; nosocomial infections
The genome sequences of three strains of Salmonella enterica subsp. enterica serovar Infantis isolated from broiler chickens in 1994 and 2004 in Hungary and in the 1980s in the United Kingdom are reported here. A sequence comparison should improve our understanding of the evolution of the genome and spread of S. Infantis in poultry.
We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid‐free serovar F isolate. Following urogenital inoculation, the plasmid‐free isolate displayed significantly reduced infectivity compared with the wild‐type strain with the latter yielding a 17‐fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid‐free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild‐type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.
Chlamydia; plasmid; mouse; virulence; infection; genome
The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity.
E. coli; O-antigen biosynthesis gene cluster; horizontal gene transfer; O serogroup; genomic diversity
Shigellosis (previously bacillary dysentery) was the primary diarrhoeal disease of World War 1, but outbreaks still occur in military operations, and shigellosis causes hundreds of thousands of deaths per year in developing nations. We aimed to generate a high-quality reference genome of the historical Shigella flexneri isolate NCTC1 and to examine the isolate for resistance to antimicrobials.
In this genomic analysis, we sequenced the oldest extant Shigella flexneri serotype 2a isolate using single-molecule real-time (SMRT) sequencing technology. Isolated from a soldier with dysentery from the British forces fighting on the Western Front in World War 1, this bacterium, NCTC1, was the first isolate accessioned into the National Collection of Type Cultures. We created a reference sequence for NCTC1, investigated the isolate for antimicrobial resistance, and undertook comparative genetics with S flexneri reference strains isolated during the 100 years since World War 1.
We discovered that NCTC1 belonged to a 2a lineage of S flexneri, with which it shares common characteristics and a large core genome. NCTC1 was resistant to penicillin and erythromycin, and contained a complement of chromosomal antimicrobial resistance genes similar to that of more recent isolates. Genomic islands gained in the S flexneri 2a lineage over time were predominately associated with additional antimicrobial resistances, virulence, and serotype conversion.
This S flexneri 2a lineage is a well adapted pathogen that has continued to respond to selective pressures. We have created a valuable historical benchmark for shigellae in the form of a high-quality reference sequence for a publicly available isolate.
The Wellcome Trust.
The results of high-resolution whole-genome sequencing data provide compelling evidence that Mycoplasma amphoriforme produces chronic relapsing infection and, importantly, is transmitted in a hospital environment.
Background. Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD). Little is known about the natural history of infection with this organism and its ability to be transmitted in the community.
Methods. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed.
Results. By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics.
Conclusions. Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen.
Mycoplasma amphoriforme; whole genome sequencing; respiratory infection; infection control; primary antibody deficiency
The “Harm Reduction” session was chaired by Dr. Jacques Normand, Director of the AIDS Research Program of the U.S. National Institute on Drug Abuse. The three presenters (and their presentation topics) were: Dr. Don Des Jarlais (High Coverage Needle/Syringe Programs for People Who Inject Drugs in Low and Middle Income Countries: A Systematic Review), Dr. Nicholas Thomson (Harm Reduction History, Response, and Current Trends in Asia), and Dr. Jih-Heng Li (Harm Reduction Strategies in Taiwan).
Harm reduction; methadone; Needle/Syringe Programs
HIV epidemics in Asia have been initially driven through injecting drug use and the use of shared needles and syringes. Molecular epidemiological work has shown that where there is heroin trafficking and use, so too is there HIV. Given the often strict enforcement of national anti-narcotic laws, harm reduction responses to HIV infections driven by injecting drug use have been historically slow. As it became clear that preventing HIV meant embracing harm reduction, many countries in the region have adopted harm reduction as part of their national AIDS strategy and increasingly as part of their national drug strategy. Initial successes have proven that harm reduction, as it pertains to HIV among IDUs, can and does work in Asia. These initial successes have led to more comprehensive scale-up of other essential components of HIV prevention among IDUs, including increased availability of opiate substitution programs. Still, multiple challenges remain as overall coverage of services in the region remains poor. Changes in the availability and patterns of use of drugs, including the exponential increase in the use of amphetamine-type stimulants, is providing ongoing challenges to both the law enforcement and public health sectors. This paper reflects on the history of harm reduction in Asia and the shifting trends forcing policy makers to adapt and expand harm reduction strategies to include an ever widening approach to criminal justice, policing, public health, and human rights.
harm reduction; HIV prevention; opiate substitution programs
Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents.
Serratia marcescens; genome plasticity; virulence; multidrug resistance
Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.
Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.
The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.
Vibrio cholerae is a diarrheal pathogen that is responsible for substantial morbidity and mortality worldwide. Historically, seven pandemics of cholera have been recognized, with classical biotype strains associated with the sixth and the El Tor biotype with the seventh (current) pandemic. Recently multi-drug resistant El Tor variants expressing classical cholera toxin have replaced the original El Tor strains in many epidemics, and are sometimes associated with more severe diarrhea leading to a higher mortality rate. In regions that experience recurrent cholera outbreaks, such as Northern India, it is important to understand the nature of the circulating strains and establish how they are related to the strains circulating globally. Here, we have demonstrated that whole- genome sequencing is a valuable method to characterize V. cholerae isolates that circulated during the 2009 outbreak in the Northern Indian city of Chandigarh. Through comparative genomic analysis, we identified two clones that circulated during a single outbreak. Importantly, these clones contain significant differences in the structure of the cholera toxin gene and the Vibrio seventh pandemic island II. Our findings demonstrate the limitations of current molecular typing techniques and the importance of surveillance with whole-genome sequencing for identifying V. cholerae clades with distinct genomic signatures.
Shigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. To define the influence of anaerobiosis on the virulence of Shigella, we performed deep RNA sequencing to identify transcriptomic differences that are induced by anaerobiosis and modulated by the anaerobic Fumarate and Nitrate Reduction regulator, FNR.
We found that 528 chromosomal genes were differentially expressed in response to anaerobic conditions; of these, 228 genes were also influenced by FNR. Genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism (e.g. ptsG, manX, murQ, cysP, cra), DNA topology and regulation (e.g. ygiP, stpA, hns), host interactions (e.g. yciD, nmpC, slyB, gapA, shf, msbB) and survival within the gastrointestinal tract (e.g. shiA, ospI, adiY, cysP). Interestingly, there was a marked effect of available oxygen on genes involved in Type III secretion system (T3SS), which is required for host cell invasion and pathogenesis. These genes, located on the large Shigella virulence plasmid, were down regulated in anaerobiosis in an FNR-dependent manner. We also confirmed anaerobic induction of csrB and csrC small RNAs in an FNR-independent manner.
Anaerobiosis promotes survival and adaption strategies of Shigella, while modulating virulence plasmid genes involved in T3SS-mediated host cell invasion. The influence of FNR on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands.
The complete genomes of two virulent phages infecting Citrobacter rodentium are reported here for the first time. Both bacteriophages were isolated from local sewage treatment plant effluents. Genome analyses revealed a close relationship between both phages and allowed their classification as members of the Autographivirinae subfamily in the T7-like genus.
Public Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined.
The most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain.
The S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive.
Shigella flexneri type strains; Next generation sequencing technology; Molecular serotyping
At least 2 subclades coexisted that had different antimicrobial-resistance profiles and patterns of spread.
In August 2010, Pakistan experienced major floods and a subsequent cholera epidemic. To clarify the population dynamics and transmission of Vibrio cholerae in Pakistan, we sequenced the genomes of all V. cholerae O1 El Tor isolates and compared the sequences to a global collection of 146 V. cholerae strains. Within the global phylogeny, all isolates from Pakistan formed 2 new subclades (PSC-1 and PSC-2), lying in the third transmission wave of the seventh-pandemic lineage that could be distinguished by signature deletions and their antimicrobial susceptibilities. Geographically, PSC-1 isolates originated from the coast, whereas PSC-2 isolates originated from inland areas flooded by the Indus River. Single-nucleotide polymorphism accumulation analysis correlated river flow direction with the spread of PSC-2. We found at least 2 sources of cholera in Pakistan during the 2010 epidemic and illustrate the value of a global genomic data bank in contextualizing cholera outbreaks.
cholera; Pakistan; whole-genome sequencing; global phylogeny; Vibrio cholerae O1; floods; bacteria; single-nucleotide polymorphism
Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004.
We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains.
Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK.
The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov).
iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.
Bacteraemia and meningitis caused by non-typhoidal Salmonella (including serovars Typhimurium, Enteritidis and Bovismorbificans) are a serious health issue in sub-Saharan Africa, particularly in young children and HIV-infected adults. Previous work has indicated that a distinct S. Typhimurium sequence type, ST313, has evolved and spread in these countries, and may be more human-adapted than isolates found in the developed world. We therefore investigated the genomes of Salmonella enterica serovar Bovismorbificans bacteraemia isolates from Malawi and compared them to genomes of veterinary S. Bovismorbificans isolates from the UK using Next Generation Sequencing Technology and subsequent genomic comparisons to establish if there is a genetic basis for this increase in invasive disease observed among African NTS. Contrary to the previous findings for S. Typhimurium, where a distinct ST is found only in sub-Saharan Africa, we discovered that the S. Bovismorbificans isolates from Malawi belong to the most common ST of the serovar and the genome is highly conserved across all sequenced isolates. The major differences between UK veterinary and African human isolates were due to prophage regions inserted into the genomes of African isolates, coupled with a higher prevalence of a virulence plasmid compared to the UK isolates.