Biological characterization of the phage lysin AVPL and its efficiency against Aerococcus viridans-induced mastitis in a murine model.

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans. IMPORTANCE: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 µg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

Aerococcus kribbianus sp. nov., a facultatively anaerobic bacterium isolated from pig faeces.

Two cocci-shaped, facultatively anaerobic, Gram-positive bacteria isolated from the faeces of a pig were designated as strains YH-aer221T and YH-aer222. Analysis of the 16S rRNA gene sequences revealed that the isolates were most closely related to Aerococcus suis JCM 18035T with 96.6 % similarity. The multi-locus sequence tree revealed that the isolates formed a sub-cluster adjacent to A. suis JCM 18035T. The average nucleotide identity values for the isolates and their most closely related strains were 71.8 and 71.7 %, respectively; and the digital DNA-DNA hybridization values for the isolates and their most closely related strains were 25.6 and 25.5 %, respectively. The main fatty acids were C18 : 1ω9c, C16 : 0 and C18 : 0. The cell wall contained the meso-diaminopimelic acid-based peptidoglycan. The two isolates shared the same metabolic pathways. Isolates YH-aer221T and YH-aer222 harboured the same CRISPR array with 33 and 46 spacers, respectively. Single-genome vs. metagenome analysis showed that the genomes of the isolates were not found in the available metagenome database. Given their chemotaxonomic, phenotypic and phylogenetic properties, YH-aer221T (= KCTC 25571T=JCM 35699T) and YH-aer222 (=KCTC 25573=JCM 35700) represent a novel taxon. The name Aerococcus kribbianus sp. nov. is proposed.

Taxonomic considerations on Aerococcus urinae with proposal of subdivision into Aerococcus urinae, Aerococcus tenax sp. nov., Aerococcus mictus sp. nov., and Aerococcus loyolae sp. nov.

Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae, an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268T = CCUG 34223T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302T = DSM 115700T = CCUG 76531T=NR-58630T), Aerococcus mictus sp. nov. (ATCC TSD-301T = DSM 115699T = CCUG 76532T=NR-58629T), and Aerococcus loyolae sp. nov. (ATCC TSD-300T = DSM 115698T = CCUG 76533T=NR-58628T) for three of the newly identified genomic taxa.

Aerococcus agrisoli sp. nov., isolated from paddy soil.

A non-spore-forming, Gram-stain-positive, short rod-shaped strain, designated SJQ22T, was isolated from a paddy soil sample collected in Shanghai, PR China. A comparative analysis of 16S rRNA gene sequences showed that strain SJQ22T fell within the genus Aerococcus, forming a clear cluster with the type strains of Aerococcus viridans (98.6 % sequence similarity) and Aerococcus urinaeequi (98.5 % sequence similarity). Strain SJQ22T grew at 30-45 °C (optimum, 30 °C), pH 6.0-8.0 (optimum, pH 7.0) and with a NaCl concentration of 0-4 % (optimum, 1 %). Cells were negative for oxidase and catalase activity. Chemotaxonomic analysis showed that strain SJQ22T possessed C16:0 and C18:1 ω9c as the predominant fatty acids. The DNA G + C content was 39.0 mol%. Strain SJQ22T exhibited DNA-DNA relatedness levels of 13±2 % with A. viridans ATCC 11563T and 9±2 % with A. urinaeequi IFO 12173T. Based on the data obtained, strain SJQ22T represents a novel species of the genus Aerococcus, for which the name Aerococcus agrisoli sp. nov. is proposed. The type strain is SJQ22T (=JCM 33111T=CCTCC AB 2018283T).

Aerococcus urinae - significance of detection in the paediatric urinary tract: a case series.

Aerococcus urinae (A. urinae) is primarily recognized as a common pathogen in the geriatric population, causing urinary tract infection (UTI), sepsis, and endocarditis, predominantly in female patients. In the paediatric population, only a few case reports exist suggesting A. urinae causes malodorous urine in otherwise healthy boys. In this study, we investigated the spectrum of clinical and laboratory presentations of A. urinae detection in children. A retrospective, single-centre, case series including all patients with the detection of A. urinae during a 7-year study period. Patients with detection of A. urinae only in non-urogenital skin swabs were excluded. A total of 40 samples from 33 patients were identified of which 20 patients were included in the final analysis. The median (IQR) age was 6.8 (2.9-9.5) years; 18 (90%) patients were boys. Four patients were diagnosed with a UTI, six had malodorous urine without UTI, three were diagnosed with balanitis and seven showed A. urinae colonization in the urine culture. Urogenital disorders were present in 12 patients. Additional pathogens were detected in 13 patients. Recurrence of detection during our study period was observed in four (20%) patients.  Conclusion: Beyond malodorous urine, A. urinae detection is associated with more severe presentations including UTI in the paediatric population. Pre-existing urogenital disorders were frequent, and therefore, a nephro-urological investigation should be considered in all cases of A. urinae detection in the paediatric population. What is Known: • Aerococcus urinae (A. urinae) is known to be a common pathogen in the geriatric population, causing urinary tract infection (UTI), sepsis, and endocarditis, predominantly in female patients. • In the paediatric population, A. urinae is mainly described as a low-grade pathogen. Some case reports describe A. urinae as the cause of extraordinary malodorous urine in otherwise healthy boys. What is New: • Beyond malodorous urine, A. urinae detection is associated with more severe presentations including UTI in the paediatric population. • A. urinae was mainly detected in boys with pre-existing urogenital disorders; therefore, a nephro-urological investigation should be considered in cases of A. urinae detection in the paediatric population.

Aerococcus viridans Phage Lysin AVPL Had Lytic Activity against Streptococcus suis in a Mouse Bacteremia Model.

Streptococcus suis (S. suis) is a swine pathogen that can cause sepsis, meningitis, endocarditis, and other infectious diseases; it is also a zoonotic pathogen that has caused a global surge in fatal human infections. The widespread prevalence of multidrug-resistant S. suis strains and the decline in novel antibiotic candidates have necessitated the development of alternative antimicrobial agents. In this study, AVPL, the Aerococcus viridans (A. viridans) phage lysin, was found to exhibit efficient bactericidal activity and broad lytic activity against multiple serotypes of S. suis. A final concentration of 300 µg/mL AVPL reduced S. suis counts by 4-4.5 log10 within 1 h in vitro. Importantly, AVPL effectively inhibited 48 h S. suis biofilm formation and disrupted preformed biofilms. In a mouse model, 300 µg/mouse AVPL protected 100% of mice from infection following the administration of lethal doses of multidrug-resistant S. suis type 2 (SS2) strain SC19, reduced the bacterial load in different organs, and effectively alleviated inflammation and histopathological damage in infected mice. These data suggest that AVPL is a valuable candidate antimicrobial agent for treating S. suis infections.

Clinical and microbiological characterization of Aerococcus urinae bacteraemias at Helsinki metropolitan area, Finland.

Our objective was to assess the incidence of bacteraemic Aerococcus urinae cases at Helsinki metropolitan area, Finland, from a 6-year study period (2013 to 2018) and to further characterize available cases. The study evaluates the outcome of commonly used cefuroxime treatment and determinate a set of A. urinae in vitro antimicrobial susceptibilities for benzylpenicillin, cefuroxime, and ceftriaxone. Clinical records of A. urinae bacteraemic patients were reviewed retrospectively. Antimicrobial susceptibility testing was performed by disk diffusion, gradient test, and broth microdilution for 139-141 clinical A. urinae isolates. Clinical data of 72/77 patients were combined with the in vitro susceptibilities. We found an increasing number of bacteraemic A. urinae cases within 6-year study period (p = 0.01). The patients were mainly elderly males, and all suffered from underlying conditions. A total of 27.3% of cases (21/77) showed polymicrobial blood cultures. Thirty-day mortality was 22.1%. Cefuroxime was the initial empiric antimicrobial agent given for 66/76 of the patients and treatment outcome was favorable for 20/22 patients who received cefuroxime at least up to day 5. All isolates were susceptible to benzylpenicillin and cefuroxime interpreted by EUCAST breakpoints for Aerococci and PK-PD breakpoints, respectively. MIC determinations gave variable results for ceftriaxone, 2.1-2.9% of the isolates were resistant. To conclude, it seems that the number of bacteraemic Aerococcus urinae cases is increasing at Helsinki metropolitan area, Finland, reflecting the growing blood culture sampling. Clinical A. urinae isolates were susceptible to cefuroxime in vitro. Treatment data indicate that empirical cefuroxime started for possibly urinary tract -derived community-acquired bacteraemia covers A. urinae.

Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5.

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Validation of a Gradient Diffusion Method (Etest) for Testing of Antimicrobial Susceptibility of Aerococcus urinae to Fluoroquinolones.

Aerococcus urinae is a urinary pathogen with well-described resistance to fluoroquinolones. This study aimed to validate the gradient diffusion (GD) method (Etest) on cation-adjusted Mueller-Hinton agar with 5% sheep blood for testing the susceptibilities of Aerococcus urinae to the antimicrobial agents ciprofloxacin and levofloxacin and to compare the Etest to the broth microdilution (BMD) method from CLSI document M45-A3. Agar dilution (AD), as recommended by EUCAST, was used as an alternative reference method to arbitrate discrepancies or address technical issues. Aerococcus urinae isolates from urinary specimens were prospectively collected between June 2016 and December 2017 from six hospitals in Quebec, Canada, and identifications were confirmed using Vitek MS with the IVD 3.0 database. Of the 207 isolates tested using BMD, 37 (17.9%) showed trailing and 19 (9.2%) showed insufficient growth; these were tested using AD. Also, 38 isolates (18.4%) for ciprofloxacin and 13 isolates (6.3%) for levofloxacin showed a lack of essential or categorical agreement between the Etest and BMD and were also tested by AD. By use of a combined reference method (BMD or AD), the susceptibility rates of Aerococcus urinae were 82.6% and 81.6% for ciprofloxacin and levofloxacin, respectively. Categorical agreement between GD and the combined reference methods was 95.2% for ciprofloxacin and 97.1% for levofloxacin, with no very major error identified. Major and minor error rates were 0.6% and 4.3% for ciprofloxacin and 1.2% and 1.9% for levofloxacin. Overall, antimicrobial susceptibility testing (AST) using the Etest on sheep blood agar showed good agreement with the reference methods and can be considered by clinical laboratories wishing to perform AST on Aerococcus urinae isolates.

Postcalving udder health and productivity in cows approaching dry-off with intramammary infections caused by non-aureus Staphylococcus, Aerococcus, Enterococcus, Lactococcus, and Streptococcus species.

The objective of this prospective cohort study was to explore associations between intramammary infection (IMI) in late-lactation cows and postcalving udder health and productivity. Cows (n = 2,763) from 74 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each visit, aseptic quarter milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status at enrollment. Cows were followed from enrollment until 120 d in milk (DIM) in the subsequent lactation. Herd records were used to establish whether subjects experienced clinical mastitis or removal from the herd, and DHIA test-day data were used to record subclinical mastitis events (somatic cell count >200,000 cells/mL) and milk yield (kg/d) during the follow-up period. Cox regression and generalized estimating equations were used to evaluate the associations between IMI and the outcome of interest. The presence of late-lactation IMI caused by major pathogens was positively associated with postcalving clinical mastitis [hazard ratio = 1.5, 95% confidence interval (CI): 1.2, 2.0] and subclinical mastitis (risk ratio = 1.5, 95% CI: 1.3, 1.9). Species within the non-aureus Staphylococcus (NAS) group varied in their associations with postcalving udder health, with some species being associated with increases in clinical and subclinical mastitis in the subsequent lactation. Late-lactation IMI caused by Streptococcus and Streptococcus (Strep)-like organisms, other than Aerococcus spp. (i.e., Enterococcus, Lactococcus, and Streptococcus spp.) were associated with increases in postcalving clinical and subclinical mastitis. Test-day milk yield from 1 to 120 DIM was lower (-0.9 kg, 95% CI: -1.6, -0.3) in late-lactation cows with any IMI compared with cows without IMI. No associations were detected between IMI in late lactation and risk for postcalving removal from the herd within the first 120 DIM. Effect estimates reported in this study may be less than the underlying quarter-level effect size for IMI at dry-off and postcalving clinical and subclinical mastitis, because of the use of late-lactation IMI as a proxy for IMI at dry-off and the use of cow-level exposure and outcome measurements. Furthermore, the large number of models run in this study (n = 94) increases the chance of identifying chance associations. Therefore, confirmatory studies should be conducted. We conclude that IMI in late lactation may increase risk of clinical and subclinical mastitis in the subsequent lactation. The relationship between IMI and postcalving health and productivity is likely to vary among pathogens, with Staphylococcus aureus, Streptococcus spp., Enterococcus spp., and Lactococcus spp. being the most important pathogens identified in the current study.

Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity.

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.

Aerococcus urinae Isolated from Women with Lower Urinary Tract Symptoms: In Vitro Aggregation and Genome Analysis.

Aerococcus urinae is increasingly recognized as a potentially significant urinary tract bacterium. A. urinae has been isolated from urine collected from both males and females with a wide range of clinical conditions, including urinary tract infection (UTI), urgency urinary incontinence (UUI), and overactive bladder (OAB). A. urinae is of particular clinical concern because it is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections. Previous genomic characterization studies have examined A. urinae strains isolated from patients experiencing UTI episodes. Here, we analyzed the genomes of A. urinae strains isolated as part of the urinary microbiome from patients with UUI or OAB. Furthermore, we report that certain A. urinae strains exhibit aggregative in vitro phenotypes, including flocking, which can be modified by various growth medium conditions. Finally, we performed in-depth genomic comparisons to identify pathways that distinguish flocking and nonflocking strains.IMPORTANCEAerococcus urinae is a urinary bacterium of emerging clinical interest. Here, we explored the ability of 24 strains of A. urinae isolated from women with lower urinary tract symptoms to display aggregation phenotypes in vitro We sequenced and analyzed the genomes of these A. urinae strains. We performed functional genomic analyses to determine whether the in vitro hyperflocking aggregation phenotype displayed by certain A. urinae strains was related to the presence or absence of certain pathways. Our findings demonstrate that A. urinae strains have different propensities to display aggregative properties in vitro and suggest a potential association between phylogeny and flocking.

Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study.

BACKGROUND: Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence. OBJECTIVE: This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome. STUDY DESIGN: With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated. RESULTS: Participants had a mean age of 53 years (range 22-90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture-detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence. CONCLUSION: In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.

Real-time neurochemical measurement of dynamic metabolic events during cardiac arrest and resuscitation in a porcine model.

This work describes a fully-integrated portable microfluidic analysis system for real-time monitoring of dynamic changes in glucose and lactate occurring in the brain as a result of cardiac arrest and resuscitation. Brain metabolites are sampled using FDA-approved microdialysis probes and coupled to a high-temporal resolution 3D printed microfluidic chip housing glucose and lactate biosensors. The microfluidic biosensors are integrated with a wireless 2-channel potentiostat forming a compact analysis system that is ideal for use in a crowded operating theatre. Data are transmitted to a custom-written app running on a tablet for real-time visualisation of metabolic trends. In a proof-of-concept porcine model of cardiac arrest, the integrated analysis system proved reliable in a challenging environment resembling a clinical setting; noise levels were found to be comparable with those seen in the lab and were not affected by major clinical interventions such as defibrillation of the heart. Using this system, we were able, for the first time, to measure changes in brain glucose and lactate levels caused by cardiac arrest and resuscitation; the system was sensitive to clinical interventions such as infusion of adrenaline. Trends suggest that cardiopulmonary resuscitation alone does not meet the high energy demands of the brain as metabolite levels only return to their values preceding cardiac arrest upon return of spontaneous circulation.

Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors.

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 µA/mM) and wide linear range (0-50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.

Portable Microfluidic Biosensing System for Real-Time Analysis of Microdialysate in Transplant Kidneys.

Currently, there is a severe shortage of donor kidneys that are fit for transplantation, due in part to a lack of adequate viability assessment tools for transplant organs. This work presents the integration of a novel wireless two-channel amperometric potentiostat with microneedle-based glucose and lactate biosensors housed in a 3D printed chip to create a microfluidic biosensing system that is genuinely portable. The wireless potentiostat transmits data via Bluetooth to an Android app running on a tablet. The whole miniaturized system is fully enclosed and can be integrated with microdialysis to allow continuous monitoring of tissue metabolite levels in real time. We have also developed a wireless portable automated calibration platform so that biosensors can be calibrated away from the laboratory and in transit. As a proof of concept, we have demonstrated the use of this portable analysis system to monitor porcine kidneys for the first time from organ retrieval, through warm ischemia, transportation on ice, right through to cold preservation and reperfusion. The portable system is robust and reliable in the challenging conditions of the abattoir and during kidney transportation and can detect clear physiological changes in the organ associated with clinical interventions.

Identification of two abundant Aerococcus urinae cell wall-anchored proteins.

Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6-24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease.

Clinical Significance of Isolates Known to Be Blood Culture Contaminants in Pediatric Patients.

Background and objectives: The objective of this study was to investigate the clinical significance of isolates from blood stream infection known to be blood culture contaminants in pediatric patients. Materials and Methods: Microbiological reports and medical records of all blood culture tests issued from 2002 to 2012 (n = 76,331) were retrospectively reviewed. Evaluation for potential contaminants were done by reviewing medical records of patients with the following isolates: coagulase-negative Staphylococcus, viridans group Streptococcus, Bacillus, Corynebacterium, Micrococcus, Aerococcus, and Proprionibacterium species. Repeated cultures with same isolates were considered as a single case. Cases were evaluated for their status as a pathogen. Results: Coagulase-negative Staphylococcus had clinical significance in 23.8% of all cases. Its rate of being a true pathogen was particularly high in patients with malignancy (43.7%). Viridans group Streptococcus showed clinical significance in 46.2% of all cases. Its rate of being a true pathogen was similar regardless of the underlying morbidity of the patient. The rate of being a true pathogens for remaining isolates was 27.7% for Bacillus and 19.0% for Corynebacterium species. Conclusions: Coagulase-negative Staphylococcus and viridans group Streptococcus isolates showed high probability of being true pathogens in the pediatric population, especially in patients with underlying malignancy.

Spondylodiscitis due to Aerococcus urinae and literature review.

INTRODUCTION: Aerococccus urinae (AU) is a pathogen mainly identified in male urinary tract infections and responsible for bacteremia and endocarditis. To the best of our knowledge, there are only five patients with osteomyelitis due to AU described in the literature. All of them had urinary tract disease or systemic conditions such as diabetes, and two were associated with an endocarditis. CASE REPORT: We described the first case of isolated spondylodiscitis without general or local predisposing condition, excepted age > 65 years.