Uropathogenic Escherichia coli population structure and antimicrobial susceptibility in Norfolk, UK.

BACKGROUND: Urinary tract infections (UTIs) are a frequent cause for visits to primary care providers. In alignment globally, uropathogenic Escherichia coli (UPEC) are the main aetiological agent for UTIs in Norfolk and are increasingly difficult to treat due to multi-drug resistance. OBJECTIVES: We set out to identify which clonal groups and resistance genes are disseminating in the community and hospitals in Norfolk, the first study of its kind for UPEC in this region. METHODS: We collected 199 clinical E. coli isolates causing UTIs in the community and hospital from the Clinical Microbiology laboratory at Norfolk and Norwich University Hospital between August 2021 and January 2022. These were whole-genome sequenced using the Illumina and MinION platforms for in silico MLST and antibiotic resistance determinant detection. RESULTS: The isolates were composed of 70 STs; 8 lineages represented 56.7% of this population: ST73, ST12, ST69, ST131, ST404, ST95, ST127 and ST1193. Importantly, primary UTI screening deemed 6.5% of isolates to be multidrug resistant (MDR), with high rates of resistance to ampicillin (52.1%) and trimethoprim (36.2%) in hospitals. Of concern is the probable clonal expansion of MDR groups ST131 and ST1193 in hospitals and community settings with chromosomally encoded blaCTX-M-15, blaOXA-1 and aac(6')-Ib-cr5. CONCLUSIONS: The burden of reported UTIs in Norfolk is largely caused by non-MDR isolates and mirrors similar UPEC studies nationally and internationally. Continually monitoring samples with consideration of sources will help reduce burden of disease.

Impact of Salmonella genome rearrangement on gene expression.

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure (GS) around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to GS identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved toward or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide-level measures of variation.

Characterization of a P1-bacteriophage-like plasmid (phage-plasmid) harbouring bla CTX-M-15 in Salmonella enterica serovar Typhi.

Antimicrobial-resistance (AMR) genes can be transferred between microbial cells via horizontal gene transfer (HGT), which involves mobile and integrative elements such as plasmids, bacteriophages, transposons, integrons and pathogenicity islands. Bacteriophages are found in abundance in the microbial world, but their role in virulence and AMR has not fully been elucidated in the Enterobacterales. With short-read sequencing paving the way to systematic high-throughput AMR gene detection, long-read sequencing technologies now enable us to establish how such genes are structurally connected into meaningful genomic units, raising questions about how they might cooperate to achieve their biological function. Here, we describe a novel ~98 kbp circular P1-bacteriophage-like plasmid termed ph681355 isolated from a clinical Salmonella enterica serovar Typhi isolate. It carries bla CTX-M-15, an IncY plasmid replicon (repY gene) and the ISEcP1 mobile element and is, to our knowledge, the first reported P1-bacteriophage-like plasmid (phage-plasmid) in S. enterica Typhi. We compared ph681355 to two previously described phage-plasmids, pSJ46 from S. enterica serovar Indiana and pMCR-1-P3 from Escherichia coli, and found high nucleotide similarity across the backbone. However, we saw low ph681355 backbone similarity to plasmid p60006 associated with the extensively drug-resistant S. enterica Typhi outbreak isolate in Pakistan, providing evidence of an alternative route for bla CTX-M-15 transmission. Our discovery highlights the importance of utilizing long-read sequencing in interrogating bacterial genomic architecture to fully understand AMR mechanisms and their clinical relevance. It also raises questions regarding how widespread bacteriophage-mediated HGT might be, suggesting that the resulting genomic plasticity might be higher than previously thought.

Effects of Aspirin for Primary Prevention in Persons with Diabetes Mellitus.

BACKGROUND: Diabetes mellitus is associated with an increased risk of cardiovascular events. Aspirin use reduces the risk of occlusive vascular events but increases the risk of bleeding; the balance of benefits and hazards for the prevention of first cardiovascular events in patients with diabetes is unclear. METHODS: We randomly assigned adults who had diabetes but no evident cardiovascular disease to receive aspirin at a dose of 100 mg daily or matching placebo. The primary efficacy outcome was the first serious vascular event (i.e., myocardial infarction, stroke or transient ischemic attack, or death from any vascular cause, excluding any confirmed intracranial hemorrhage). The primary safety outcome was the first major bleeding event (i.e., intracranial hemorrhage, sight-threatening bleeding event in the eye, gastrointestinal bleeding, or other serious bleeding). Secondary outcomes included gastrointestinal tract cancer. RESULTS: A total of 15,480 participants underwent randomization. During a mean follow-up of 7.4 years, serious vascular events occurred in a significantly lower percentage of participants in the aspirin group than in the placebo group (658 participants [8.5%] vs. 743 [9.6%]; rate ratio, 0.88; 95% confidence interval [CI], 0.79 to 0.97; P=0.01). In contrast, major bleeding events occurred in 314 participants (4.1%) in the aspirin group, as compared with 245 (3.2%) in the placebo group (rate ratio, 1.29; 95% CI, 1.09 to 1.52; P=0.003), with most of the excess being gastrointestinal bleeding and other extracranial bleeding. There was no significant difference between the aspirin group and the placebo group in the incidence of gastrointestinal tract cancer (157 participants [2.0%] and 158 [2.0%], respectively) or all cancers (897 [11.6%] and 887 [11.5%]); long-term follow-up for these outcomes is planned. CONCLUSIONS: Aspirin use prevented serious vascular events in persons who had diabetes and no evident cardiovascular disease at trial entry, but it also caused major bleeding events. The absolute benefits were largely counterbalanced by the bleeding hazard. (Funded by the British Heart Foundation and others; ASCEND Current Controlled Trials number, ISRCTN60635500 ; ClinicalTrials.gov number, NCT00135226 .).

Effects of n-3 Fatty Acid Supplements in Diabetes Mellitus.

BACKGROUND: Increased intake of n-3 fatty acids has been associated with a reduced risk of cardiovascular disease in observational studies, but this finding has not been confirmed in randomized trials. It remains unclear whether n-3 (also called omega-3) fatty acid supplementation has cardiovascular benefit in patients with diabetes mellitus. METHODS: We randomly assigned 15,480 patients with diabetes but without evidence of atherosclerotic cardiovascular disease to receive 1-g capsules containing either n-3 fatty acids (fatty acid group) or matching placebo (olive oil) daily. The primary outcome was a first serious vascular event (i.e., nonfatal myocardial infarction or stroke, transient ischemic attack, or vascular death, excluding confirmed intracranial hemorrhage). The secondary outcome was a first serious vascular event or any arterial revascularization. RESULTS: During a mean follow-up of 7.4 years (adherence rate, 76%), a serious vascular event occurred in 689 patients (8.9%) in the fatty acid group and in 712 (9.2%) in the placebo group (rate ratio, 0.97; 95% confidence interval [CI], 0.87 to 1.08; P=0.55). The composite outcome of a serious vascular event or revascularization occurred in 882 patients (11.4%) and 887 patients (11.5%), respectively (rate ratio, 1.00; 95% CI, 0.91 to 1.09). Death from any cause occurred in 752 patients (9.7%) in the fatty acid group and in 788 (10.2%) in the placebo group (rate ratio, 0.95; 95% CI, 0.86 to 1.05). There were no significant between-group differences in the rates of nonfatal serious adverse events. CONCLUSIONS: Among patients with diabetes without evidence of cardiovascular disease, there was no significant difference in the risk of serious vascular events between those who were assigned to receive n-3 fatty acid supplementation and those who were assigned to receive placebo. (Funded by the British Heart Foundation and others; Current Controlled Trials number, ISRCTN60635500 ; ClinicalTrials.gov number, NCT00135226 .).

A mixed-method investigation of patient monitoring and enhanced feedback in routine practice: Barriers and facilitators.

OBJECTIVE: To investigate the barriers and facilitators of an effective implementation of an outcome monitoring and feedback system in a UK National Health Service psychological therapy service. METHOD: An outcome monitoring system was introduced in two services. Enhanced feedback was given to therapists after session 4. Qualitative and quantitative methods were used, including questionnaires for therapists and patients. Thematic analysis was carried out on written and verbal feedback from therapists. Analysis of patient outcomes for 202 episodes of therapy was compared with benchmark data of 136 episodes of therapy for which feedback was not given to therapists. RESULTS: Themes influencing the feasibility and acceptability of the feedback system were the extent to which therapists integrated the measures and feedback into the therapy, availability of administrative support, information technology, and complexity of the service. There were low levels of therapist actions resulting from the feedback, including discussing the feedback in supervision and with patients. CONCLUSIONS: The findings support the feasibility and acceptability of setting up a routine system in a complex service, but a number of challenges and barriers have to be overcome and therapist differences are apparent. More research on implementation and effectiveness is needed in diverse clinical settings.

Repertoire of HLA-DR1-restricted CD4 T-cell responses to capsular Caf1 antigen of Yersinia pestis in human leukocyte antigen transgenic mice.

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in "humanized" HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens.

We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.

Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice.

Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-L-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10(3) median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.