Study within a trial of electronic versus paper-based Patient-Reported oUtcomes CollEction (SPRUCE): study protocol for a partially randomised patient preference study.

INTRODUCTION: Patient-reported outcomes (PRO) are currently collected from trial participants using paper questionnaires by the Clinical Trials and Statistics Unit at The Institute of Cancer Research (ICR-CTSU). Streamlining PRO collection using electronic questionnaires (ePRO) may improve data collection and patient experience. Here, we outline our protocol for a Study within a trial of electronic versus paper-based Patient-Reported oUtcomes CollEction (SPRUCE), which investigates the acceptability of ePRO in oncology clinical trials. METHODS AND ANALYSIS: SPRUCE was developed alongside patient and public contributors. SPRUCE runs in multiple host trials with a partially randomised patient preference design, allowing participants to be randomised or choose their preference of electronic or paper questionnaires. Questionnaires are scheduled in accordance with host trial follow-up. The primary objective will assess differences in return rates (compliance) between ePRO and paper PROs at the first timepoint post-host trial intervention in the randomised group. Paper PRO compliance is expected to be 90%. 244 randomised participants are required to exclude ≤80% compliance rates with ePRO (10% non-inferiority margin, with 80% power and one-sided alpha=0.05). SPRUCE aims to assess acceptability of ePRO in oncology clinical trials, establish whether ePRO is acceptable to ICR-CTSU trial participants and can capture complete PRO data, consistent with paper PROs. ETHICS AND DISSEMINATION: The SPRUCE protocol (ICR-CTSU/2021/10074) was approved by the Coventry and Warwick Central Research Ethics Committee (21/WM/0223) on 21 October 2021. Results will be disseminated via presentations, publications and lay summaries. No participant identifiable data will be included. TRIAL REGISTRATION: SWAT169.

Polymers Use as Mulch Films in Agriculture-A Review of History, Problems and Current Trends.

The application of mulch films for preserving soil moisture and preventing weed growth has been a part of agricultural practice for decades. Different materials have been used as mulch films, but polyethylene plastic has been considered most effective due to its excellent mechanical strength, low cost and ability to act as a barrier for sunlight and water. However, its use carries a risk of plastic pollution and health hazards, hence new laws have been passed to replace it completely with other materials over the next few years. Research to find out about new biodegradable polymers for this purpose has gained impetus in the past few years, driven by regulations and the United Nations Organization's Sustainable Development Goals. The primary requisite for these polymers is biodegradability under natural climatic conditions without the production of any toxic residual compounds. Therefore, biodegradable polymers developed from fossil fuels, microorganisms, animals and plants are viable options for using as mulching material. However, the solution is not as simple since each polymer has different mechanical properties and a compromise has to be made in terms of strength, cost and biodegradability of the polymer for its use as mulch film. This review discusses the history of mulching materials, the gradual evolution in the choice of materials, the process of biodegradation of mulch films, the regulations passed regarding material to be used, types of polymers that can be explored as potential mulch films and the future prospects in the area.

Genomic epidemiology of clinical Campylobacter spp. at a single health trust site.

Campylobacter is the leading cause of bacterial enteritis in the developed world, and infections with the organism are largely sporadic in nature. Links between sporadic cases have not been established, with the majority of infections thought to be caused by genetically distinct isolates. Using a read-mapping approach, 158 clinical isolates collected during 2014 from the greater Nottinghamshire area were analysed to assess the local population structure and investigate potential case linkages between sporadic cases of campylobacteriosis. Four instances (2.5 %) of case linkage were observed across the dataset. This study demonstrates that case linkage does occur between sporadic Campylobacter infections, and provides evidence that a dual multi-locus sequence typing/within-lineage single nucleotide polymorphism typing approach to Campylobacter genomic epidemiology provides a benefit to public-health investigations.

Genetic import and phenotype specific alleles associated with hyper-invasion in Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a major zoonotic pathogen, causing gastroenteritis in humans. Invasion is an important pathogenesis trait by which C. jejuni causes disease. Here we report the genomic analysis of 134 strains to identify traits unique to hyperinvasive isolates. METHODS: A total of 134 C. jejuni genomes were used to create a phylogenetic tree to position the hyperinvasive strains. Comparative genomics lead to the identification of mosaic capsule regions. A pan genome approach led to the discovery of unique loci, or loci with unique alleles, to the hyperinvasive strains. RESULTS: Phylogenetic analysis showed that the hyper-invasive phenotype is a generalist trait. Despite the fact that hyperinvasive strains are only distantly related based on the whole genome phylogeny, they all possess genes within the capsule region with high identity to capsule genes from C. jejuni subsp. doylei and C. lari. In addition there were genes unique to the hyper-invasive strains with identity to non-C. jejuni genes, as well as allelic variants of mainly pathogenesis related genes already known in the other C. jejuni. In particular, the sequence of flagella genes, flgD-E and flgL were highly conserved amongst the hyper-invasive strains and divergent from sequences in other C. jejuni. A novel cytolethal distending toxin (cdt) operon was also identified as present in all hyper-invasive strains in addition to the classic cdt operon present in other C. jejuni. CONCLUSIONS: Overall, the hyper-invasive phenotype is strongly linked to the presence of orthologous genes from other Campylobacter species in their genomes, notably within the capsule region, in addition to the observed association with unique allelic variants in flagellar genes and the secondary cdt operon which is unlikely under random sharing of accessory alleles in separate lineages.

Gene Loss and Lineage-Specific Restriction-Modification Systems Associated with Niche Differentiation in the Campylobacter jejuni Sequence Type 403 Clonal Complex.

Campylobacter jejuni is a highly diverse species of bacteria commonly associated with infectious intestinal disease of humans and zoonotic carriage in poultry, cattle, pigs, and other animals. The species contains a large number of distinct clonal complexes that vary from host generalist lineages commonly found in poultry, livestock, and human disease cases to host-adapted specialized lineages primarily associated with livestock or poultry. Here, we present novel data on the ST403 clonal complex of C. jejuni, a lineage that has not been reported in avian hosts. Our data show that the lineage exhibits a distinctive pattern of intralineage recombination that is accompanied by the presence of lineage-specific restriction-modification systems. Furthermore, we show that the ST403 complex has undergone gene decay at a number of loci. Our data provide a putative link between the lack of association with avian hosts of C. jejuni ST403 and both gene gain and gene loss through nonsense mutations in coding sequences of genes, resulting in pseudogene formation.

Cj1136 is required for lipooligosaccharide biosynthesis, hyperinvasion, and chick colonization by Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasive C. jejuni strains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue of cj1136, which encodes a putative galactosyltransferase according to the annotation of the C. jejuni NCTC11168 genome. In the current study, we investigated the role of cj1136 in C. jejuni virulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. The cj1136 mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation of cj1136 resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. The cj1136 mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded by cj1136 is involved in LOS biosynthesis and is important for C. jejuni virulence, as disruption of this gene and the resultant truncation of LOS affect both colonization in vivo and invasiveness in vitro.

Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings.

OBJECTIVES: To assess the molecular epidemiology and prevalence of antibiotic resistance in Escherichia coli causing urinary tract infections of elderly patients from community and hospital settings. Also, to determine whether the possession of antibiotic resistance and virulence-associated genes can be linked to patient location or the clonal group of the organisms in question. METHODS: E. coli were isolated from the urine samples of elderly patients from the Nottingham area, and subjected to antibiotic susceptibility testing, virulence gene detection by PCR and multilocus sequence typing. RESULTS: No correlation was observed between community- or hospital-derived strains with regard to antibiotic resistance levels or virulence gene profiles. E. coli ST131 (where ST stands for sequence type) was the predominant ST found in both hospital and community samples, and demonstrated high levels of antibiotic resistance to the test panel, but did not possess a significantly larger array of virulence genes or a specific gene profile compared with other STs. CONCLUSIONS: The level of antibiotic resistance or virulence gene possession in uropathogenic E. coli is not directly associated with the healthcare setting of the patient, but there is a variation in antibiotic resistance and virulence gene possession depending on clonal group. ST131 is highly virulent and demonstrates high levels of antibiotic resistance, but its virulence does not appear to be attributable to the possession of a specific virulence-associated gene set or the possession of any virulence-associated gene in significantly higher levels than in any other ST.

Increased human pathogenic potential of Escherichia coli from polymicrobial urinary tract infections in comparison to isolates from monomicrobial culture samples.

The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tract infections (UTIs) in clinical laboratories does not report bacteria isolated from samples containing three or more different bacterial species. As a result many UTIs go unreported and untreated, particularly in elderly patients, where polymicrobial UTI samples are especially prevalent. This study reports the presence of the major uropathogenic species in mixed culture urine samples from elderly patients, and of resistance to front-line antibiotics, with potentially increased levels of resistance to ciprofloxacin and trimethoprim. Most importantly, the study highlights that Escherichia coli present in polymicrobial UTI samples are statistically more invasive (P<0.001) in in vitro epithelial cell infection assays than those isolated from monomicrobial culture samples. In summary, the results of this study suggest that the current diagnostic standard procedure for polymicrobial UTI samples needs to be reassessed, and that E. coli present in polymicrobial UTI samples may pose an increased risk to human health.

Genomic characterization of Campylobacter jejuni strain M1.

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1.

Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion.

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (approximately 14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes.

BACKGROUND: The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East. RESULTS: This scheme uses 7 loci; atpD, fusA, glnS, gltB, gyrB, infB, and pps. There were 12 sequence types (ST) identified in C. sakazakii, and 3 in C. malonaticus. A third (22/60) of C. sakazakii strains were in ST4, which had almost equal numbers of clinical and infant formula isolates from 1951 to 2008. ST8 may represent a particularly virulent grouping of C. sakazakii as 7/8 strains were clinical in origin which had been isolated between 1977 - 2006, from four countries. C. malonaticus divided into three STs. The previous Cronobacter biotyping scheme did not clearly correspond with STs nor with species. CONCLUSION: In conclusion, MLST is a more robust means of identifying and discriminating between C. sakazakii and C. malonaticus than biotyping. The MLST database for these organisms is available online at http://pubmlst.org/cronobacter/.

Identification of hyperinvasive Campylobacter jejuni strains isolated from poultry and human clinical sources.

Campylobacter jejuni causes gastroenteritis with a variety of symptoms in humans. In the absence of a suitable animal model, in vitro models have been used to study virulence traits such as invasion and toxin production. In this study, 113 C. jejuni isolates from poultry and poultry-related (n=74) environments as well as isolates from human cases (n=39) of campylobacteriosis and bacteraemia were tested for invasiveness using INT 407 cells. The method was sufficiently reproducible to observe a spectrum of invasiveness amongst strains. As a result, strains were classified as low, high and hyper-invasive. The majority of strains (poultry and human) were low invaders (82 % and 88 %, respectively). High invasion was found for 5 % of human strains and 11 % of poultry-related isolates. However, only 1 % of poultry strains were classified as hyperinvasive compared to 13 % of human isolates (P=0.0182). Of those isolates derived from the blood of bacteraemic patients, 20 % were hyperinvasive, though this correlation was not statistically significant. An attempt was made to correlate invasiveness with the presence of seven genes previously reported to be associated with virulence. Most of these genes did not correlate with invasiveness, but gene cj0486 was weakly over-represented, and a negative correlation was observed for the gene ciaB. This trend was stronger when the two genes were analysed together, thus ciaB(-) cj0486(+) was over-represented in high and hyperinvasive strains, with low invaders more commonly found to lack these genes (P=0.0064).

Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni.

The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.

Host-associated genetic import in Campylobacter jejuni.

Host association of Campylobacter jejuni was analyzed by using multilocus sequence typing data for 713 isolates from chickens and bovids (cattle and sheep). Commonly used summary measures of genotypes (sequence type and clonal complex) showed poor accuracy, but a method using the full allelic profile showed 80% accuracy in distinguishing isolates from these 2 host groups. We explored the biologic basis of more accurate results with allelic profiles. Strains isolated from specific hosts have imported a substantial number of alleles while circulating in those host species. These results imply that 1) although Campylobacter moves frequently between hosts, most transmission is within species, and 2) lineages can acquire a host signature and potentially adapt to the host through recombination. Assignment using this signature enables improved prediction of source for pathogens that undergo frequent genetic recombination.

An aflagellate mutant Yersinia enterocolitica biotype 1A strain displays altered invasion of epithelial cells, persistence in macrophages, and cytokine secretion profiles in vitro.

Despite being classically defined as non-pathogenic, there is growing evidence that biotype 1A Yersinia enterocolitica isolates may be aetiological agents of disease in humans. In previous studies, a potential link between motility and the ability of biotype 1A strains to invade cultured epithelial cells was observed. In an attempt to further investigate this finding, a flagella mutant was constructed in a human faecal Y. enterocolitica biotype 1A isolate. The flagella mutation abolished the ability of the strain to invade cultured human epithelial cells, although adherence was not affected. The aflagellate mutant was also attenuated in its ability to survive within cultured macrophages, being cleared after 3 h, whilst the wild-type persisted for 24 h after infection. Examination of cytokine secretion by infected macrophages also suggested that the flagella of biotype 1A strains act as anti-inflammatory agents, decreasing production of tumour necrosis factor (TNF)-alpha whilst increasing secretion of interleukin (IL)-10. Preliminary studies using porcine in vitro organ culture (IVOC) tissue suggested that the flagella mutant was also attenuated in its ability to colonize intestinal tissue.

Yersinia enterocolitica isolates of differing biotypes from humans and animals are adherent, invasive and persist in macrophages, but differ in cytokine secretion profiles in vitro.

Previous epidemiological studies have demonstrated a potential link between the serotypes of Yersinia enterocolitica recovered from cattle, sheep and pigs and those isolated from human disease cases. Further studies utilizing amplified fragment length polymorphisms have shown a relationship at the genetic level between strains of biotypes 3 and 4 from humans and livestock, and also suggested that some biotype 1A isolates, classically defined as non-pathogenic, are closely related to biotype 3 and 4 isolates. This study sought to understand further the pathogenic potential of Y. enterocolitica isolates from livestock in Great Britain. A range of surrogate in vitro models, such as invasion of epithelial tissue cultures, survival in cultured macrophages and cytokine secretion response, was employed to assess the pathogenicity of 88 strains. The results suggested that all isolates examined were capable of adhering to and invading epithelial cells and of surviving within macrophages. However, the inflammatory response of the infected macrophages differed with the infecting Y. enterocolitica subtype, with the response to pathogenic biotype 3 and 4 isolates different to that observed with biotype 1A isolates, and with the biotype 3 O : 5,27 isolates recovered exclusively from animals. Infections of porcine tissue also suggested the possibility of host-tissue tropism within Y. enterocolitica subtypes.

Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica.

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype.

Cytolethal distending toxin (CDT)-negative Campylobacter jejuni strains and anti-CDT neutralizing antibodies are induced during human infection but not during colonization in chickens.

The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulence-associated phenotypes.

The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods [corrected].