Molecular methods to investigate adhesion, transmigration, invasion and intracellular survival of the foodborne pathogen Campylobacter jejuni

AbstractCampylobacter jejuni is a spiral-shaped Gram-negative pathogen and major agent of gastrointestinal foodborne illness in humans worldwide. This pathogen encodes numerous described pathogenicity-associated factors involved in important processes including bacterial adhesion to, transmigration across, invasion into and intracellular survival within intestinal epithelial cells. This review article highlights various molecular techniques applied in the studies of each of these individual steps of C. jejuni host cell interactions in vitro including gentamicin protection assay, chemotaxis and motility assays, transwell and intracellular survival assays, G-Lisa, siRNA knockdown, immunohistochemistry, immunofluorescence, electron microscopy and luciferase reporter assays. We discuss the strengths and limitations of the methods as well as the different cell model systems applied. Future work should employ new technologies including modern microscopic, proteomics-based and cell signaling approaches to identify and characterise novel virulence mechanisms, which are crucial to provide fresh insights into the diversity of strategies employed by this important pathogen to cause disease

Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni

Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni

Mosaic DNA imports with interspersions of recipient sequence after natural transformation of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen

Structural organisation of the type IV secretion systems

Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNAs and/or proteins through the membranes of bacteria. Because of their complexity and multi-protein organisation, T4S systems have been extremely challenging to study structurally. However in the past five years significant milestones have been achieved by X-ray crystallography and cryo-electron microscopy. This review describes some of the more recent advances: the structures of some of the protein components of the T4S systems and the complete core complex structure that was determined using electron microscopy

Targeted high-throughput sequencing of tagged nucleic acid samples

High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa

BackgroundThe awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent.MethodologyWe searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients.FindingsA total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections.ConclusionThe small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies

Communication and patient safety in gynecology and obstetrics - study protocol of an intervention study.

BACKGROUND: Patient safety is a key target in public health, health services and medicine. Communication between all parties involved in gynecology and obstetrics (clinical staff/professionals, expectant mothers/patients and their partners, close relatives or friends providing social support) should be improved to ensure patient safety, including the avoidance of preventable adverse events (pAEs). Therefore, interventions including an app will be developed in this project through a participatory approach integrating two theoretical models. The interventions will be designed to support participants in their communication with each other and to overcome difficulties in everyday hospital life. The aim is to foster effective communication in order to reduce the frequency of pAEs. If communication is improved, clinical staff should show an increase in work satisfaction and patients should show an increase in patient satisfaction. METHODS: The study will take place in two maternity clinics in Germany. In line with previous studies of complex interventions, it is divided into three interdependent phases. Each phase provides its own methods and data. Phase 1: Needs assessment and a training for staff (n = 140) tested in a pre-experimental study with a pre/post-design. Phase 2: Assessment of communication training for patients and their social support providers (n = 423) in a randomized controlled study. Phase 3: Assessment of an app supporting the communication between staff, patients, and their social support providers (n = 423) in a case-control study. The primary outcome is improvement of communication competencies. A range of other implementation outcomes will also be assessed (i.e. pAEs, patient/treatment satisfaction, work satisfaction, safety culture, training-related outcomes). DISCUSSION: This is the first large intervention study on communication and patient safety in gynecology and obstetrics integrating two theoretical models that have not been applied to this setting. It is expected that the interventions, including the app, will improve communication practice which is linked to a lower probability of pAEs. The app will offer an effective and inexpensive way to promote effective communication independent of users' motivation. Insights gained from this study can inform other patient safety interventions and health policy developments. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03855735; date of registration: February 27, 2019

Host-associated Genetic Import in Campylobacter jejuni

C. jejuni genomes have a host signature that enables attribution of isolates to animal sources

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. Results: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. Conclusions: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments

Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950–1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus