Transcriptomic and proteomic analysis of the virulence inducing effect of ciprofloxacin on enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is considered to be the most dangerous pathotype of E. coli, as it causes severe conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Antibiotic treatment of EHEC infections is generally not recommended since it may promote the production of the Shiga toxin (Stx) and lead to worsened symptoms. This study explores how exposure to the fluoroquinolone ciprofloxacin reorganizes the transcriptome and proteome of EHEC O157:H7 strain EDL933, with special emphasis on virulence-associated factors. As expected, exposure to ciprofloxacin caused an extensive upregulation of SOS-response- and Stx-phage proteins, including Stx. A range of other virulence-associated factors were also upregulated, including many genes encoded by the LEE-pathogenicity island, the enterohemolysin gene (ehxA), as well as several genes and proteins involved in LPS production. However, a large proportion of the genes and proteins (17 and 8%, respectively) whose expression was upregulated upon ciprofloxacin exposure (17 and 8%, respectively) are not functionally assigned. This indicates a knowledge gap in our understanding of mechanisms involved in EHECs response to antibiotic-induced stress. Altogether, the results contribute to better understanding of how exposure to ciprofloxacin influences the virulome of EHEC and generates a knowledge base for further studies on how EHEC responds to antibiotic-induced stress. A deeper understanding on how EHEC responds to antibiotics will facilitate development of novel and safer treatments for EHEC infections.

Invasive meningococcal disease in Norway in the two decades before the COVID-19 pandemic.

OBJECTIVES: Disease caused by the bacterium Neisseria meningitidis remains a worldwide public health challenge, despite the steadily decreasing incidence in Western countries. The objective of this study was to explore the epidemiology of invasive meningococcal disease in Norway over the last two decades. DESIGN: All isolates sent to the National Reference Laboratory from patients with invasive meningococcal disease between the years 2000 and 2019 were analyzed using whole genome sequencing (total: 625). RESULTS: A five-fold decrease in case numbers occurred over this period, and the situation has gone from being dominated by serogroup B to one where serogroups Y and W are more prevalent. Concurrently, the mean age at infection has increased from 18 to 33 years. Among the 350 serogroup B isolates, 87% were an exact match or cross-reactive with one or both the currently available serogroup B vaccines, but the proportion decreased in the past decade. Core genome analyses revealed a high variation in the number of allelic differences accumulated in epidemiologically linked isolates to the point that near-identical isolates were found several years apart. CONCLUSION: Allelic distance is an imprecise metric for the degree of epidemiologic linkage between isolates in N. meningitidis.

Comprehensive Contact Tracing, Testing and Sequencing Show Limited Transmission of SARS-CoV-2 between Children in Schools in Norway, August 2020 to May 2021.

The role of children in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in schools has been a topic of controversy. In this study among school contacts of SARS-CoV-2 positive children in 43 contact-investigations, we investigated SARS-CoV-2 transmission in Norway, August 2020-May 2021. All participants were tested twice within seven to ten days, using SARS-CoV-2 PCR on home-sampled saliva. Positive samples were whole genome sequenced. Among the 559 child contacts, eight tested positive (1.4%, 95% CI 0.62-2.80), with no significant difference between primary (1.0%, 95% CI 0.27-2.53) and secondary schools (2.6%, 95% CI 0.70-6.39), p = 0.229, nor by viral strain, non-Alpha (1.4%, 95% CI 0.50-2.94) and Alpha variant (B.1.1.7) (1.7%, 95% CI 0.21-5.99), p = 0.665. One adult contact (1/100) tested positive. In 34 index cases, we detected 13 different SARS-CoV-2 Pango lineage variants, with B.1.1.7 being most frequent. In the eight contact-investigations with SARS-CoV-2 positive contacts, four had the same sequence identity as the index, one had no relation, and three were inconclusive. With mitigation measures in place, the spread of SARS-CoV-2 from children in schools is limited. By excluding contact-investigations with adult cases known at the time of enrolment, our data provide a valid estimate on the role of children in the transmission of SARS-CoV-2 in schools.

Genomic epidemiology and population structure of Neisseria gonorrhoeae in Norway, 2016-2017.

This study presents the nationwide epidemiology of Neisseria gonorrhoeae, using whole-genome sequencing of all culture-positive cases, which comprise roughly 40 % of all cases of gonorrhea reported in Norway from 2016 to 2017. Isolates were assigned to sequence types and Bayesian analysis clusters and variation in genes coding for antibiotic resistance was linked to phenotypic resistance data. The study also included isolates taken from the same patients from different anatomical sites at one or more time points. Comparing these isolates allows for observation of patterns of infections, i.e. multiple reinfections of genetically related clones vs. reinfections of genetically distant clones, and quantification of the genomic variation of closely related isolates from samples taken from a patient within the same day. Demographically, the patients in the study could be split into two groups; one group of patients from the capital with a high proportion of men who have sex with men (MSM), and another consisting of young adults with transmission primarily between males and females from outside the capital. Some clusters of N. gonorrhoeae were restricted to one of these two demographic groups. Pairwise comparison of multiple isolates from the same patients revealed that most were reinfected with different clones. Observations of frequent reinfections in patients is a concern and should be taken into account in the development of improved information and treatment guidelines.

Gauging the epidemic potential of a widely circulating non-invasive meningococcal strain in Africa.

Neisseria meningitidis colonizes the human oropharynx and transmits mainly via asymptomatic carriage. Actual outbreaks of meningococcal meningitis are comparatively rare and occur when susceptible populations are exposed to hypervirulent clones, genetically distinct from the main carriage isolates. However, carriage isolates can evolve into pathogens through a limited number of recombination events. The present study examines the potential for the sequence type (ST)-192, by far the dominant clone recovered in recent meningococcal carriage studies in sub-Saharan Africa, to evolve into a pathogen. We used whole-genome sequencing on a collection of 478 meningococcal isolates sampled from 1- to 29- year-old healthy individuals in Arba Minch, southern Ethiopia in 2014. The ST-192 clone was identified in nearly 60 % of the carriers. Using complementary short- and long-read techniques for whole-genome sequencing, we were able to completely resolve genomes and thereby identify genomic differences between the ST-192 carriage strain and known pathogenic clones with the highest possible resolution. We conclude that it is possible, but unlikely, that ST-192 could evolve into a significant pathogen, thus, becoming the major invasive meningococcus clone in the meningitis belt of Africa following upcoming mass vaccination with a polyvalent conjugate vaccine that targets the A, C, W, Y and X capsules.

Pancreas disease caused by Salmonid alphavirus subtype 2 reduces growth and feed conversion in farmed Atlantic salmon.

Pancreas disease (PD), caused by several subtypes of salmonid alphavirus (SAV), is associated with significant economic losses in European salmonid aquaculture. In this retrospective cohort study, we investigate the impact of PD caused by SAV subtype 2 (SAV2) on growth, feed conversion, and mortality in farmed Atlantic salmon (Salmo salar L.). The study was based on harvest data from a large salmon farming company operating in the SAV2 endemic area of Norway. Mixed-effect regression analyses showed a severe impact on both growth and feed conversion when PD appeared late in the production cycle. In a scenario with fixed slaughter time the estimated impact corresponded to a growth reduction of 0.7 kg and 0.07 points increase in feed conversion ratio. No effect on mortality was observed in this data set. In conclusion, the most important consequences of PD caused by SAV2 infection is reduced growth and feed conversion in large Atlantic salmon. The lack of effect on mortality in this study may be due to other factors overshadowing the impact of PD.

Phylogenetic relationships and regional spread of meningococcal strains in the meningitis belt, 2011-2016.

BACKGROUND: Historically, the major cause of meningococcal epidemics in the meningitis belt of sub-Saharan Africa has been Neisseria meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine starting in 2010. We performed whole-genome sequencing on isolates collected post-2010 to assess their phylogenetic relationships and inter-country transmission. METHODS: A total of 716 invasive meningococcal isolates collected between 2011 and 2016 from 11 meningitis belt countries were whole-genome sequenced for molecular characterization by the three WHO Collaborating Centers for Meningitis. FINDINGS: We identified three previously-reported clonal complexes (CC): CC11 (n = 434), CC181 (n = 62) and CC5 (n = 90) primarily associated with NmW, NmX, and NmA, respectively, and an emerging CC10217 (n = 126) associated with NmC. CC11 expanded throughout the meningitis belt independent of the 2000 Hajj outbreak strain, with isolates from Central African countries forming a distinct sub-lineage within this expansion. Two major sub-lineages were identified for CC181 isolates, one mainly expanding in West African countries and the other found in Chad. CC10217 isolates from the large outbreaks in Nigeria and Niger were more closely related than those from the few cases in Mali and Burkina Faso. INTERPRETATIONS: Whole-genome based phylogenies revealed geographically distinct strain circulation as well as inter-country transmission events. Our results stress the importance of continued meningococcal molecular surveillance in the region, as well as the development of an affordable vaccine targeting these strains. FUND: Meningitis Research Foundation; CDC's Office of Advanced Molecular Detection; GAVI, the Vaccine Alliance.

A genomic view of experimental intraspecies and interspecies transformation of a rifampicin-resistance allele into Neisseria meningitidis.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.

Acquisition of virulence genes by a carrier strain gave rise to the ongoing epidemics of meningococcal disease in West Africa.

In the African meningitis belt, a region of sub-Saharan Africa comprising 22 countries from Senegal in the west to Ethiopia in the east, large epidemics of serogroup A meningococcal meningitis have occurred periodically. After gradual introduction from 2010 of mass vaccination with a monovalent meningococcal A conjugate vaccine, serogroup A epidemics have been eliminated. Starting in 2013, the northwestern part of Nigeria has been affected by yearly outbreaks of meningitis caused by a novel strain of serogroup C Neisseria meningitidis (NmC). In 2015, the strain spread to the neighboring country Niger, where it caused a severe epidemic. Following a relative calm in 2016, the largest ever recorded epidemic of NmC broke out in Nigeria in 2017. Here, we describe the recent evolution of this new outbreak strain and show how the acquisition of capsule genes and virulence factors by a strain previously circulating asymptomatically in the African population led to the emergence of a virulent pathogen. This study illustrates the power of long-read whole-genome sequencing, combined with Illumina sequencing, for high-resolution epidemiological investigations.

Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates.

Species within the genus Neisseria display significant glycan diversity associated with the O-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail the pgl genotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. The O-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All major pgl locus polymorphisms identified in Neisseria meningitidis to date were present in our isolate collection, with the variable presence of pglG and pglH, both in combination with either pglB or pglB2 We identified significant changes and diversity in the pgl genotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and the O-acetyltransferase. To our knowledge, this is the first characterization of the pgl genotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity in N. meningitidis and into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described in Neisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

Whole genome sequencing reveals within-host genetic changes in paired meningococcal carriage isolates from Ethiopia.

BACKGROUND: Meningococcal colonization is a prerequisite for transmission and disease, but the bacterium only very infrequently causes disease while asymptomatic carriage is common. Carriage is highly dynamic, showing a great variety across time and space within and across populations, but also within individuals. The understanding of genetic changes in the meningococcus during carriage, when the bacteria resides in its natural niche, is important for understanding not only the carriage state, but the dynamics of the entire meningococcal population. RESULTS: Paired meningococcal isolates, obtained from 50 asymptomatic carriers about 2 months apart were analyzed with whole genome sequencing (WGS). Phylogenetic analysis revealed that most paired isolates from the same individual were closely related, and the average and median number of allelic differences between paired isolates defined as the same strain was 35. About twice as many differences were seen between isolates from different individuals within the same sequence type (ST). In 8%, different strains were detected at different time points. A difference in ST was observed in 6%, including an individual who was found to carry three different STs over the course of 9 weeks. One individual carried different strains from the same ST. In total, 566 of 1605 cgMLST genes had undergone within-host genetic changes in one or more pairs. The most frequently changed cgMLST gene was relA that was changed in 47% of pairs. Across the whole genome, pilE, differed mostly, in 85% of the pairs. The most frequent mechanisms of genetic difference between paired isolates were phase variation and recombination, including gene conversion. Different STs showed variation with regard to which genes that were most frequently changed, mostly due to absence/presence of phase variation. CONCLUSIONS: This study revealed within-host genetic differences in meningococcal isolates during short-term asymptomatic carriage. The most frequently changed genes were genes belonging to the pilin family, the restriction/modification system, opacity proteins and genes involved in glycosylation. Higher resolution genome-wide sequence typing is necessary to resolve the diversity of isolates and reveals genetic differences not discovered by traditional typing schemes, and would be the preferred choice of technology.

Whole-Genome Characterization of Epidemic Neisseria meningitidis Serogroup C and Resurgence of Serogroup W, Niger, 2015.

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013-2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.

Peri-pubertal gonadotropin-releasing hormone agonist treatment affects sex biased gene expression of amygdala in sheep.

The nature of hormonal involvement in pubertal brain development has attracted wide interest. Structural changes within the brain that occur during pubertal development appear mainly in regions closely linked with emotion, motivation and cognitive functions. Using a sheep model, we have previously shown that peri-pubertal pharmacological blockade of gonadotropin releasing hormone (GnRH) receptors, results in exaggerated sex-differences in cognitive executive function and emotional control, as well as sex and hemisphere specific patterns of expression of hippocampal genes associated with synaptic plasticity and endocrine signaling. In this study, we explored effects of this treatment regime on the gene expression profile of the ovine amygdala. The study was conducted with 30 same-sex twin lambs (14 female and 16 male), half of which were treated with the GnRH agonist (GnRHa) goserelin acetate every 4th week, beginning before puberty, until approximately 50 weeks of age. Gene expression profiles of the left and right amygdala were measured using 8×15 K Agilent ovine microarrays. Differential expression of selected genes was confirmed by qRT-PCR (Quantitative real time PCR). Networking analyses and Gene Ontology (GO) Term analyses were performed with Ingenuity Pathway Analysis (IPA), version 7.5 and DAVID (Database for Annotation, Visualization and integrated Discovery) version 6.7 software packages, respectively. GnRHa treatment was associated with significant sex- and hemisphere-specific differential patterns of gene expression. GnRHa treatment was associated with differential expression of 432 (|logFC|>0.3, adj. p value <0.05) and 46 (p value <0.0.5) genes in the left and right amygdala, respectively, of female animals, relative to the reference sample which consisted of all a pooled sample from control and treated animals of both sexes. No genes were found to be differentially expressed as a result of GnRHa treatment in the male animals. The results indicated that GnRH may, directly and/or indirectly, be involved in the regulation of sex- and hemisphere-specific differential expression of genes in the amygdala. This finding should be considered when long-term peri-pubertal GnRHa treatment is used in children.