Impact of growth media and pressure on the diversity and antimicrobial activity of isolates from two species of hexactinellid sponge.

Access to deep-sea sponges brings with it the potential to discover novel antimicrobial candidates, as well as novel cold- and pressure-adapted bacteria with further potential clinical or industrial applications. In this study, we implemented a combination of different growth media, increased pressure and high-throughput techniques to optimize recovery of isolates from two deep-sea hexactinellid sponges, Pheronema carpenteri and Hertwigia sp., in the first culture-based microbial analysis of these two sponges. Using 16S rRNA gene sequencing for isolate identification, we found a similar number of cultivable taxa from each sponge species, as well as improved recovery of morphotypes from P. carpenteri at 22-25 °C compared to other temperatures, which allows a greater potential for screening for novel antimicrobial compounds. Bacteria recovered under conditions of increased pressure were from the phyla Proteobacteria, Actinobacteria and Firmicutes, except at 4 %O2/5 bar, when the phylum Firmicutes was not observed. Cultured isolates from both sponge species displayed antimicrobial activity against Micrococcus luteus, Staphylococcus aureus and Escherichia coli.

The Phylogenetic Structure of Reptile, Avian and Uropathogenic Escherichia coli with Particular Reference to Extraintestinal Pathotypes.

The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).

Dual transcriptional-translational cascade permits cellular level tuneable expression control.

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.

Comprehensive analysis of type 1 fimbriae regulation in fimB-null strains from the multidrug resistant Escherichia coli ST131 clone.

Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC.

A single dose of epidermicin NI01 is sufficient to eradicate MRSA from the nares of cotton rats.

Objectives: To investigate the efficacy of a potent novel antimicrobial protein of mass 6 kDa, epidermicin NI01, for eradicating the nasal burden of MRSA in a cotton rat ( Sigmodon hispidus ) model. Methods: MRSA strain ATCC 43300 was used to establish a robust colonization of cotton rat nares. This model was used to evaluate the efficacy of topical 0.04% and 0.2% epidermicin NI01, administered twice daily for 3 days consecutively, and topical 0.8% epidermicin NI01 administered once, for reducing nasal MRSA burden. Control groups remained untreated or were administered vehicle only (0.5% hydroxypropylmethylcellulose) or 2% mupirocin twice daily for 3 days. The experiment was terminated at day 5 and MRSA quantitative counts were determined. Tissues recovered from animals treated with 0.2% epidermicin twice daily for 3 days were examined for histological changes. Results: Mupirocin treatment resulted in a reduction in burden of log 10 (log R) of 2.59 cfu/nares compared with vehicle ( P < 0.0001). Epidermicin NI01 administered once at 0.8% showed excellent efficacy, resulting in a log R of 2.10 cfu/nares ( P = 0.0004), which was equivalent to mupirocin. Epidermicin NI01 administered at 0.2% or 0.04% twice daily for 3 days did not have a significant impact on the tissue burden recovered from the nares. Mild to marked histological abnormalities were noted, but these were determined to be reversible. Conclusion: A single dose of topical epidermicin NI01 was as effective as mupirocin administered twice daily for 3 days in eradication of MRSA from the nares of cotton rats. This justifies further development of epidermicin for this indication.

Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform.

BACKGROUND: The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols.. METHODS: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME. RESULTS: There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms). CONCLUSIONS: The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.

Global dissemination of a multidrug resistant Escherichia coli clone.

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum ß-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

The skin microbiome in psoriatic arthritis: methodology development and pilot data.

BACKGROUND: Skin microbiota are likely to be important in the development of conditions such as psoriatic arthritis. Profiling the bacterial community in the psosriatic plaques will contribute to our understanding of the role of the skin microbiome in these conditions. The aim of this work was to determine the optimum study design for work on the skin microbiome with use of the MiSeq platform. The objectives were to compare data generated from two platforms for two primer pairs in a low density mock bacterial community. METHODS: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of four healthy volunteers. The DNA was amplified with primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3), and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 platforms and sequenced. Both datasets were de-noised, cleaned of chimeras, and analysed by use of QIIME software (version 1.8.0). FINDINGS: No significant difference in the diversity indices at the phylum and the genus level between the platforms was seen. Comparison of the diversity indices for the mock community data for the two primer pairs demonstrated that the V3-V4 hypervariable region had significantly better capture of bacterial diversity than did the V1-V3 region. Amplification with the same primer pairs showed strong concordance within each platform (98·9-99·8%), with negligible effect of spiked human DNA contamination. Comparison at the family level classification between samples processed on the MiSeq and Roche454 platforms using the V3-V4 hypervariable region also showed a high level of concordance (87%), although less so for the V1-V3 primers (10%). The pilot data from healthy volunteers were similar. INTERPRETATION: Results obtained from the V3-V4 16S rRNA hypervariable region, sequencing on the MiSeq and Roche454 platforms, were concordant between replicates, and between each other. These findings suggest that the MiSeq platform, and these primers, is a comparable method for determining skin microbiota to the widely used Roche454 methodology. FUNDING: NIHR Manchester Musculoskeletal Biomedical Research Unit.

Domestic shower hose biofilms contain fungal species capable of causing opportunistic infection.

The domestic environment can be a source of pathogenic bacteria. We show here that domestic shower hoses may harbour potentially pathogenic bacteria and fungi. Well-developed biofilms were physically removed from the internal surface of shower hoses collected in four locations in England and Scotland. Amplicon pyrosequencing of 16S and 18S rRNA targets revealed the presence of common aquatic and environmental bacteria, including members of the Actinobacteria, Alphaproteobacteria, Bacteroidetes and non-tuberculous Mycobacteria. These bacteria are associated with infections in immunocompromised hosts and are widely reported in shower systems and as causes of water-acquired infection. More importantly, this study represents the first detailed analysis of fungal populations in shower systems and revealed the presence of sequences related to Exophiala mesophila, Fusarium fujikuroi and Malassezia restricta. These organisms can be associated with the environment and healthy skin, but also with infection in compromised and immuno-competent hosts and occurrence of dandruff. Domestic showering may result in exposure to aerosols of bacteria and fungi that are potentially pathogenic and toxigenic. It may be prudent to limit development of these biofilms by the use of disinfectants, or regular replacement of hoses, where immuno-compromised persons are present.

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Antibiotic-resistant ST38, ST131 and ST405 strains are the leading uropathogenic Escherichia coli clones in Riyadh, Saudi Arabia.

OBJECTIVES: We investigated the molecular epidemiology of uropathogenic Escherichia coli (UPEC) from a tertiary care hospital in Riyadh, Saudi Arabia, revealing, for the first time, the population structure of UPEC in the region. METHODS: A total of 202 UPEC isolates were recovered from hospital and community patients with urinary tract infection in December 2012 and January 2013. Strains were characterized by MLST, antibiotic susceptibility determination and virulence gene detection. RESULTS: The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%), ST10 (6.4%), ST127 (5.9%), ST95 (5.4%), ST12 (3.5%), ST998 (3.5%) and ST405 (3%). ST131 and ST405 isolates were significantly associated with high levels of antibiotic resistance (60% of ST131 carried CTX-M-14 or CTX-M-15 and 66.7% of ST405 isolates carried CTX-M-15). ST131, CTX-M-15-positive isolates were predominantly of the fimH30/clade C group, resistant to fluoroquinolones; members of this sub-group were more likely to carry a high number of genes encoding selected virulence determinants. The relatively high proportion of ST38 was notable and four of these isolates harboured aggR. CONCLUSIONS: Our findings highlight the presence of MDR, CTX-M-positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion of isolates with CTX-M is a particular concern. We suggest that ST38 UPEC warrant further study.

Third-generation cephalosporin resistance conferred by a chromosomally encoded blaCMY-23 gene in the Escherichia coli ST131 reference strain EC958.

OBJECTIVES: Escherichia coli ST131 is a globally disseminated MDR clone originally identified due to its association with the blaCTX-M-15 gene encoding an ESBL. It is thus assumed that blaCTX-M-15 is the major determinant for resistance to ß-lactam antibiotics in this clone. The complete sequence of EC958, a reference strain for E. coli ST131, revealed that it contains a chromosomally located blaCMY-23 gene with an upstream ISEcp1 element as well as several additional plasmid-encoded ß-lactamase genes. Here, we examined the genetic context of the blaCMY-23 element in EC958 and other E. coli ST131 strains and investigated the contribution of blaCMY-23 to EC958 resistance to a range of ß-lactam antibiotics. METHODS: The genetic context of blaCMY-23 and its associated mobile elements was determined by PCR and sequencing. Antibiotic susceptibility testing was performed using Etests. The activity of the blaCMY-23 promoter was assessed using lacZ reporter assays. Mutations were generated using λ-Red-recombination. RESULTS: The genetic structure of the ISEcp1-IS5-blaCMY-23 mobile element was determined and localized within the betU gene on the chromosome of EC958 and five other E. coli ST131 strains. The transcription of blaCMY-23, driven by a previously defined promoter within ISEcp1, was significantly higher than other ß-lactamase genes and could be induced by cefotaxime. Deletion of the blaCMY-23 gene resulted in enhanced susceptibility to cefoxitin, cefotaxime and ceftazidime. CONCLUSIONS: This is the first known report to demonstrate the chromosomal location of blaCMY-23 in E. coli ST131. In EC958, CMY-23 plays a major role in resistance to third-generation cephalosporins and cephamycins.

The bacterial skin microbiome in psoriatic arthritis, an unexplored link in pathogenesis: challenges and opportunities offered by recent technological advances.

The resident microbial community, harboured by humans in sites such as the skin and gastrointestinal tract, is enormous, representing a candidate environmental factor affecting susceptibility to complex diseases, where both genetic and environmental risk factors are important. The potential of microorganisms to influence the human immune system is considerable, given their ubiquity. The impact of the host-gene-microbe interaction on the maintenance of health and the development of disease has not yet been assessed robustly in chronic inflammatory conditions. PsA represents a model inflammatory disease to explore the role of the microbiome because skin involvement and overlap with IBD implicates both the skin and gastrointestinal tract as sources of microbial triggers for PsA. In parallel with genetic studies, characterization of the host microbiota may benefit our understanding of the microbial contribution to disease pathogenesis-knowledge that may eventually inform the development of novel therapeutics.

Multiple metabolomics of uropathogenic E. coli reveal different information content in terms of metabolic potential compared to virulence factors.

No single analytical method can cover the whole metabolome and the choice of which platform to use may inadvertently introduce chemical selectivity. In order to investigate this we analysed a collection of uropathogenic Escherichia coli. The selected strains had previously undergone extensive characterisation using classical microbiological methods for a variety of metabolic tests and virulence factors. These bacteria were analysed using Fourier transform infrared (FT-IR) spectroscopy; gas chromatography mass spectrometry (GC-MS) after derivatisation of polar non-volatile analytes; as well as reversed-phase liquid chromatography mass spectrometry in both positive (LC-MS(+ve)) and negative (LC-MS(-ve)) electrospray ionisation modes. A comparison of the discriminatory ability of these four methods with the metabolic test and virulence factors was made using Procrustes transformations to ascertain which methods produce congruent results. We found that FT-IR and LC-MS(-ve), but not LC-MS(+ve), were comparable with each other and gave highly similar clustering compared with the virulence factors tests. By contrast, FT-IR and LC-MS(-ve) were not comparable to the metabolic tests, and we found that the GC-MS profiles were significantly more congruent with the metabolic tests than the virulence determinants. We conclude that metabolomics investigations may be biased to the analytical platform that is used and reflects the chemistry employed by the methods. We therefore consider that multiple platforms should be employed where possible and that the analyst should consider that there is a danger of false correlations between the analytical data and the biological characteristics of interest if the full metabolome has not been measured.

A FimH inhibitor prevents acute bladder infection and treats chronic cystitis caused by multidrug-resistant uropathogenic Escherichia coli ST131.

BACKGROUND: Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent. METHODS: Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958. RESULTS: We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958-mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold. CONCLUSIONS: In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli.

Non-allergist healthcare workers views on delivering a penicillin allergy de-labelling inpatient pathway: identifying the barriers and enablers.

Background: Non-allergist delivered PADL is supported by UK and World Health Organization guidelines but is not yet routine in UK hospitals. Understanding the views of healthcare workers (HCWs) on managing patients with penA records and exploring perspectives on delivering a PADL inpatient pathway are required to inform the development of non-allergist delivered PADL pathways. Objective: To explore the perspectives of non-allergist HCWs working in medical specialties on managing patients with penA records, and to explore the enablers and barriers to embedding PADL as a standard of care for inpatients. Methods: Semi-structured interviews with doctors, nurses, pharmacists and medicines optimization pharmacy technicians working in a district general hospital in the UK. Thematic analysis was used to analyse the data. Results: The PADL pathway was considered a shared responsibility of the multidisciplinary team, which needed to be structured and supported by a framework. PADL aligns with HCW roles but time to deliver PADL was a barrier. Training for HCWs on the benefits of PADL and delivering PADL for those patients where a penicillin might be beneficial during the current episode of care would both motivate HCWs to deliver PADL. Discussion and conclusion: The PADL pathway was acceptable to HCWs and aligned with their roles and current healthcare processes but their capacity to deliver PADL in a time pressured environment was a significant barrier.

Experiences of an inpatient penicillin allergy de-labelling pathway: capturing the patient voice.

Background: Non-allergist-delivered penicillin allergy de-labelling (PADL) is supported by UK and other national guidelines but is not yet routine practice in UK hospitals. Those who have undergone PADL report high rates of acceptance, but it is unknown why some continue to avoid penicillin, and why some decline testing. Objectives: To explore the experiences of patients recently approached for penicillin allergy (penA) assessment and de-label by non-allergists in a UK hospital to determine the barriers and enablers to patient acceptance of PADL. Methods: Qualitative study using semi-structured interviews with patients who were penA assessed and de-labelled during an inpatient stay between November 2022 and January 2023. Thematic analysis was used to analyse the data. Results: Nineteen patients were interviewed. Patients were largely unaware of the negative impact of penA on their healthcare. Patients had differing views on challenging their penA status while they were acutely unwell, some agreeing that it is the right time to test and others not. Patients declined testing because they felt they were at higher potential risk because they were older or had multiple comorbidities. Some patients who declined testing felt they would have been persuaded if they had received a better explanation of the risks and benefits of PADL. Conclusions: Patients who were successfully de-labelled were positive about the experience. Those who declined testing did so for a variety of reasons including frailty/comorbidities or a fear of testing whilst unwell. Patients highlighted the importance of good communication about the personalized risks and benefits of testing.

Immunogenicity and Protective Capacity of Sugar ABC Transporter Substrate-Binding Protein against Streptococcus suis Serotype 2, 7 and 9 Infection in Mice.

Background:Streptococcus suis (S. suis) is a Gram-positive bacterium that causes substantial disease in pigs. S. suis is also an emerging zoonoses in humans, primarily in Asia, through the consumption of undercooked pork and the handling of infected pig meat as well as carcasses. The complexity of S. suis epidemiology, characterized by the presence of multiple bacterial serotypes and strains with diverse sequence types, identifies a critical need for a universal vaccine with the ability to confer cross-protective immunity. Highly conserved immunogenic proteins are generally considered good candidate antigens for subunit universal vaccines. Methods: In this study, the cross-protection of the sugar ABC transporter substrate-binding protein (S-ABC), a surface-associated immunogenic protein of S. suis, was examined in mice for evaluation as a universal vaccine candidate. Results: S-ABC was shown to be highly conserved, with 97% amino acid sequence identity across 31 S. suis strains deposited in GenBank. Recombinantly expressed S-ABC (rS-ABC) was recognized via rabbit sera specific to S. suis serotype 2. The immunization of mice with rS-ABC induced antigen-specific antibody responses, as well as IFN-γ and IL-4, in multiple organs, including the lungs. rS-ABC immunization conferred high (87.5% and 100%) protection against challenges with S. suis serotypes 2 and 9, demonstrating high cross-protection against these serotypes. Protection, albeit lower (50%), was also observed in mice challenged with S. suis serotype 7. Conclusions: These data identify S-ABC as a promising antigenic target within a universal subunit vaccine against S. suis.

International multicenter evaluation of the DiversiLab bacterial typing system for Escherichia coli and Klebsiella spp.

Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.