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).

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.

Identification of an Aerococcus urinaeequi isolate by whole genome sequencing and average nucleotide identity analysis.

OBJECTIVES: Identification and classification of microorganisms is one of the most important but difficult and challenging issues in microbiology. Whole genome sequencing (WGS), which can give a thorough understanding for the genome of bacteria strain, has been universally used for studying bacterial classification, evolution, and drug-related resistant genes. We in this study aimed to identify a Gram-positive, microaerophilic, catalase-negative cocci strain named AV208, which has shown resistance to vancomycin, by whole genome's average nucleotide identity (ANI) and high-throughput sequencing technology. METHODS: The AV208 strain was identified by following commercially available identification systems, including API 20 Strep system and Vitek 2 Compact Gram-positive identification system for biochemical phenotypic test. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and 16S rRNA gene sequencing were used for confirmation identification. The whole genome of AV208 was sequenced by using high throughput sequencing technology and ANI between AV208, and its phylogenetic neighbours were analysed by the Orthologous Average Nucleotide Identity Tool (OAT) software. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the potential molecular mechanism for vancomycin resistance. RESULTS: The AV208 strain was isolated from an ascites sample from a patient with chronic kidney disease who showed extensive resistance to the drugs detected, such as vancomycin with MIC >256 µg/mL. With combination of biochemical phenotypic test, MALDI-TOF-MS and 16S rRNA gene sequencing, the AV208 strain was tentatively identified as an Aercoccus viridans. By using complete genome sequence, we found a 96.24% ANI between strain AV208 and Aerococcus urinaeequi CCUG 28094T, which was higher than that with A. viridans CCUG4311T (94.9%). The consistency of 16S rRNA sequence of strain AV208 was 100% with A. urinaeequi CCUG 28094T and 99.9% with A. viridans CCUG4311T, with only one base difference between them. PCR and sequencing for van genes revealed that AV208 was positive for the vanA gene. A Tn1546 transposon-like structure with vanA gene was found in the genome, which was predicted locating in plasmid, causing vancomycin resistance phenotypes. CONCLUSION: Average nucleotide identity analysis based on whole genome sequence is an accurate and effective method for identification of bacteria, especially for strains that are not discernible by existing methods such as Aerococcus.

A mouse model displays host and bacterial strain differences in Aerococcus urinae urinary tract infection.

In recent years, the clinical significance of Aerococcus urinae has been increasingly recognized. A. urinae has been implicated in cases of urinary tract infection (UTI; acute cystitis and pyelonephritis) in both male and female patients, ranging from children to older adults. Aerococcus urinae can also be invasive, causing urosepsis, endocarditis, and musculoskeletal infections. Mechanisms of pathogenesis in A. urinae infections are poorly understood, largely due to the lack of an animal model system. In response to this gap, we developed a model of A. urinae urinary tract infection in mice. We compared A. urinae UTI in female C3H/HeN and C57BL/6 mice and compared four clinical isolates of A. urinae isolated from patients with UTI, urgency urinary incontinence, and overactive bladder. Our data demonstrate that host genetic background modulates A. urinae UTI. Female C57BL/6 female mice rapidly cleared the infection. Female C3H/HeN mice, which have inherent vesicoureteral reflux that flushes urine from the bladder up into the kidneys, were susceptible to prolonged bacteriuria. This result is consistent with the fact that A. urinae infections most frequently occur in patients with underlying urinary tract abnormalities or disorders that make them susceptible to bacterial infection. Unlike uropathogens such as E. coli, which cause infection and inflammation both of the bladder and kidneys in C3H/HeN mice, A. urinae displayed tropism for the kidney, persisting in kidney tissue even after clearance of bacteria from the bladder. Aerococcus urinae strains from different genetic clades displayed varying propensities to cause persistent kidney infection. Aerococcus urinae infected kidneys displayed histological inflammation, neutrophil recruitment and increased pro-inflammatory cytokines. These results set the stage for future research that interrogates host-pathogen interactions between A. urinae and the urinary tract.

Background of patients with Aerococcus spp. detected in blood cultures, and drug sensitivity of Aerococcus spp.

This study aimed to review the clinical characteristics of patients with Aerococcus spp. detected by blood culture, and drug susceptibility of Aerococcus spp. All cases of Aerococcus spp. determined using blood culture between June 2013 and May 2020 in a single institution were included; patient information (age, sex, comorbidities, outcome, diagnosis, antimicrobial agents) was analyzed. The cohort comprised 25 patients (18 [72%] men and 7 [28%] women; median age, 84.5 [range, 75-87] years). Thirteen (52%) patients had urinary tract infections(UTI) caused by Aerococcus spp. All patients had a favorable prognosis, except 1 who died owing to infective endocarditis. Drug susceptibility testing showed that most isolates were susceptible to ß-lactams except 1. However, 24 (96%) cases were resistant to trimethoprim-sulfamethoxazole and 10 (40%) to quinolones. Aerococcus spp. are important causative agents of bacteremia and UTI. The increasing reports of Aerococcus spp. infections could lead to better treatment schemes and facilitate diagnosis.

A novel plasmid from Aerococcus urinaeequi of porcine origin co-harboring the tetracycline resistance genes tet(58) and tet(61).

Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the presence of two different tet genes conferring different resistance mechanisms on the same plasmid has rarely been reported. In this study, we identified the tandem tetracycline resistance genes tet(61)-tet(58) on the novel plasmid pT4303. These tet genes were identified for the first time in Aerococcus urinaeequi. Reduced susceptibility to doxycycline was observed in S. aureus RN4220 harboring tet(61) when an extra tet(58) was expressed. Plasmid pT4303 was electrotransformed into S. aureus RN4220, E. faecalis JH2-2, S. suis BAA and E. coli DH5α and conferred tetracycline resistance (MIC ≥ 16) in both Gram-positive and Gram-negative bacteria, assuming that it might serve as a vehicle for the dissemination of the tetracycline resistance genes tet(61) and tet(58).

Characteristics of Aerococcus viridans isolated from porcine fetuses in Korean farms.

Swine abortion caused by viruses as well as bacteria has caused many economic losses in domestic farms over the years; however, bacterial abortion has not yet been studied in Korea. Several bacterial species were isolated from aborted fetuses (n = 103) for which the cause of death was not viral abortion. Among them, we focused on Aerococcus viridans, which had the highest positive rate within three provinces (Gangwon, Jeonnam and Gyeongnam). A total of 16 isolates were identified as A. viridans by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and 13 were characterized by both antibiotic resistance and 16S rRNA gene analysis. Based on antibiotic susceptibility testing result, eight antimicrobials could not effectively eliminate the present isolation (more than 40% of isolates can resist these antibiotics), while all except two strains were susceptible to trimethoprim/sulfamethoxazole. Molecular analysis indicated genetic variation among these strains. This study is the first report detecting A. viridans from aborted fetuses in Korean domestic farms.

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.

Purification and Characterization of an Antibacterial Substance from Aerococcus urinaeequi Strain HS36.

A bacterial strain inhibiting the growth of Vibrio anguillarum, the causative agent of vibriosis, was isolated from fish intestines. The isolated strain HS36 was identified as Aerococcus urinaeequi based on the characteristics of the genus according to Bergey's Manual of Systematic Bacteriology and by 16S rRNA sequencing. The growth rate and antibacterial activity of strain HS36 in shaking culture were higher than those in static culture, while the optimal pH and temperature for antibacterial activity were 7.0 and 30°C, respectively. The active antibacterial substance was purified from a culture broth of A. urinaeequi HS36 by Sephadex G-75 gel chromatography, Sephadex G-25 gel chromatography, and reverse-phase high-performance liquid chromatography. Its molecular weight, as estimated by Tricine SDS-polyacrylamide gel electrophoresis, was approximately 1,000 Da. The antibacterial substance produced by strain HS36 was stable after incubation for 1 h at 100°C. Although its antibacterial activity was optimal at pH 6-8, activity was retained at a pH range from 2 to 11. The purified antibacterial substance was inactivated by proteinase K, papain, and ß-amylase treatment. The newly purified antibacterial substance, classified as a class II bacteriocin, inhibited the growth of Klebsiella pneumoniae, Salmonella enterica, and Vibrio alginolyticus.

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.

High-level expression of Aerococcus viridans pyruvate oxidase in Escherichia coli by optimization of vectors and induction conditions.

Pyruvate oxidase is an important enzyme used as a reagent in kits and biochemical analyses; however, the yield of pyruvate oxidase from wild microbial strains is low. In this study, high-level expression of Aerococcus viridans pyruvate oxidase was achieved in recombinant Escherichia coli by optimizing the expression system and induction conditions. Three recombinant pET vectors were constructed for pyruvate oxidase expression in E. coli. The isopropyl-ß-d-thiogalactoside (IPTG) concentration and induction temperature were optimized, with the result that the highest pyruvate oxidase yield (4106·9 U l-1 ) of the recombinant E. colipET28a-pod was obtained under conditions of 25°C, 0·5 mmol l-1 IPTG, 0·5 OD600 , after 24 h of induction, which was 34·2 times the yield achieved with the wild-type strain. The soluble pyruvate oxidase contributed 99·6% of the total pyruvate oxidase expressed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a highly soluble pyruvate oxidase can be obtained in recombinant Escherichia coli by optimizing vectors and induction conditions. The pyruvate oxidase yield achieved is the highest reported so far, which provides a convenient and cost-saving way to produce pyruvate oxidase. This research promotes pyruvate oxidase application in the pharmaceutical and biochemical industries.

Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli.

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD600) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26 hr fermentation.

Genomic characterization, phylogenetic analysis, and identification of virulence factors in Aerococcus sanguinicola and Aerococcus urinae strains isolated from infection episodes.

Aerococcus sanguinicola and Aerococcus urinae are emerging pathogens in clinical settings mostly being causative agents of urinary tract infections (UTIs), urogenic sepsis and more seldomly complicated infective endocarditis (IE). Limited knowledge exists concerning the pathogenicity of these two species. Eight clinical A. sanguinicola (isolated from 2009 to 2015) and 40 clinical A. urinae (isolated from 1984 to 2015) strains from episodes of UTIs, bacteremia, and IE were whole-genome sequenced (WGS) to analyze genomic diversity and characterization of virulence genes involved in the bacterial pathogenicity. A. sanguinicola genome sizes were 2.06-2.12 Mb with 47.4-47.6% GC-contents, and 1783-1905 genes were predicted whereof 1170 were core-genes. In case of A. urinae strains, the genome sizes were 1.93-2.44 Mb with 41.6-42.6% GC-contents, and 1708-2256 genes of which 907 were core-genes. Marked differences were observed within A. urinae strains with respect to the average genome sizes, number and sequence identity of core-genes, proteome conservations, phylogenetic analysis, and putative capsular polysaccharide (CPS) loci sequences. Strains of A. sanguinicola showed high degree of homology. Phylogenetic analyses showed the 40 A. urinae strains formed two clusters according to two time periods: 1984-2004 strains and 2010-2015 strains. Genes that were homologs to virulence genes associated with bacterial adhesion and antiphagocytosis were identified by aligning A. sanguinicola and A. urinae pan- and core-genes against Virulence Factors of Bacterial Pathogens (VFDB). Bacterial adherence associated gene homologs were present in genomes of A. sanguinicola (htpB, fbpA, lmb, and ilpA) and A. urinae (htpB, lap, lmb, fbp54, and ilpA). Fifteen and 11-16 CPS gene homologs were identified in genomes of A. sanguinicola and A. urinae strains, respectively. Analysis of these genes identified one type of putative CPS locus within all A. sanguinicola strains. In A. urinae genomes, five different CPS loci types were identified with variations in CPS locus sizes, genetic content, and structural organization. In conclusion, this is the first study dealing with WGS and comparative genomics of clinical A. sanguinicola and A. urinae strains from episodes of UTIs, bacteremia, and IE. Gene homologs associated with antiphagocytosis and bacterial adherence were identified and genetic variability was observed within A. urinae genomes. These findings contribute with important knowledge and basis for future molecular and experimental pathogenicity study of UTIs, bacteremia, and IE causing A. sanguinicola and A. urinae strains.

An initially unidentified case of urinary tract infection due to Aerococcus urinae.

Aerococcus urinae is a microorganism responsible for urinary tract and blood stream infections which are rarely reported in clinical practice. However, it has been proposed that the infrequency of such reports may be partially due to difficulties related to pathogen identification. We present here a case of an elderly male patient with urinary tract infection where A. urinae was initially not identified by a private microbiology laboratory. Our report highlights the need to consider A. urinae as a causative agent of urinary tract infections because if not identified and properly treated it may lead to endocarditis or septicemia.

The Gestational Vaginal Microbiome and Spontaneous Preterm Birth among Nulliparous African American Women.

Introduction Early markers to identify pregnant women at high risk for spontaneous preterm birth (SPTB) have not been established and preventive options are limited. Recent attention has focused on examining the importance of characterizing the vaginal microbiome to predict SPTB. Results We examined the diversity and structure of the vaginal microbiome in nulliparous African American women during early pregnancy and compared 13 women who delivered preterm and 27 women who delivered at term. Samples were taken at one of two points in gestation, before 16 weeks or between 20 and 24 weeks. Among women who delivered preterm, we found lower bacterial diversity with lower abundance of Coriobacteriaceae, Sneathia, Prevotella, and Aerococcus compared with women delivering at term (linear discriminant analysis score > 3.0). The Shannon diversity index was not significantly different between the groups (p-value = 0.239). Phylogenetic diversity and Chao1 suggested a lower diversity in the vaginal microbiota of women who delivered preterm compared with term, but these findings were not significantly different (p = 0.077 and p = 0.066, respectively). Conclusion These data suggest that the vaginal microbiome of women delivering preterm had lower diversity than women delivering after 37 weeks, although these findings need to be explored in a larger sample of nulliparous African American women.

A Case of Sepsis in a 92-Year-Old Korean Woman Caused by Aerococcus urinae and Identified by Sequencing the 16S Ribosomal RNA Gene.

Aerococcus urinae is an uncommon pathogen that was first identified in 1992. Herein, we report a case of bloodstream infection caused by A. urinae, which occurred in a 92-year-old Korean female patient with an underlying urologic infection who had altered consciousness. The blood culture yielded positive results for A. urinae; however, identifying A. urinae was challenging. Ultimately, we used 16S ribosomal RNA (rRNA) gene sequencing to identify the organism. The patient recovered after being treated with ertapenem and meropenem. To our knowledge, this is the first report of a case of A. urinae sepsis in South Korea.

Molecular and antibiotic susceptibility characterization of Aerococcus viridans isolated from porcine urinary infection.

Aerococcus viridans has been reported as a human and animal pathogen causing urinary tract infection, arthritis, pneumonia, meningitis and endocarditis. Routinely, A. viridans is not surveyed in clinical diagnosis laboratories and commonly is misidentified as other bacteria. There is no concrete data on the prevalence and impact of the pathogen to both human and animal health. In the present study, we report the isolation and molecular and antibiotic susceptibility characterization of A. viridans strains from porcine urinary infections. A total of 22 isolates were identified as A. viridans by MALDI-TOF MS and confirmed by 16S rRNA gene sequencing. Isolates were genotyped by single enzyme amplified fragments length polymorphism (SE-AFLP) that resulted in 19 clusters of which 81.2% were composed by single isolates. The high genetic heterogeneity corroborates previous studies and appears to be a particularity of A. viridans. The minimal inhibitory concentration (MIC) values also presented variability especially for ceftiofur, fluoroquinolones and aminoglycosides. The high MICs of aminoglycosides, tetracyclines and macrolides seen among the A. viridans corroborate previous reports and the widespread veterinary usage of these antibiotics demand attention for the implication of A. viridans infection to both human and animal health.