Expanding the Bacterial Diversity of the Female Urinary Microbiome: Description of Eight New Corynebacterium Species.

The genus Corynebacterium is frequently found in the female urinary microbiome (FUM). In-depth characterization of Corynebacterium at the species level has been barely exploited. During ongoing FUM research studies, eight strains (c8Ua_144T, c8Ua_172T, c8Ua_174T, c8Ua_181T, c9Ua_112T, c19Ua_109T, c19Ua_121T, and c21Ua_68T) isolated from urine samples of healthy women or diagnosed with overactive bladder could not be allocated to any valid Corynebacterium species. In this work, we aimed to characterize these strains based on a polyphasic approach. The strains were Gram stain positive, rod to coccoid shaped, nonmotile, catalase positive, and oxidase negative. Phylogenetic analysis based on 16S rRNA and rpoB gene sequences indicated that all strains belonged to the genus Corynebacterium. The average nucleotide identity and digital DNA-DNA hybridization values among the genomes of the above eight strains and closely related type strains of the Corynebacterium genus were <95 (74.1%-93.9%) and <70% (22.2%-56.5%), respectively. Mycolic acids were identified in all strains. MK-8(H2) and/or MK-9(H2) were identified as the major menaquinones. The polar lipids' pattern mostly consisted of diphosphatidylglycerol, phosphatidylglycerol, and glycophospholipids. The major fatty acid was C18:1ω9c. Corynebacterium lehmanniae (c8Ua_144T = DSM 113405T = CCP 74T), Corynebacterium meitnerae (c8Ua_172T = DSM 113406T = CCP 75T), Corynebacterium evansiae (c8Ua_174T = DSM 113407T = CCP 76T), Corynebacterium curieae (c8Ua_181T = DSM 113408T = CCP 77T), Corynebacterium macclintockiae (c9Ua_112T = DSM 113409T = CCP 78T), Corynebacterium hesseae (c19Ua_109T = DSM 113410T= CCP 79T), Corynebacterium marquesiae (c19Ua_121T = DSM 113411T = CCP 80T), and Corynebacterium yonathiae (c21Ua_68T = DSM 113412T = CCP 81T) are proposed. This study evidenced that commonly used methodologies on FUM research presented limited resolution for discriminating Corynebacterium at the species level. Future research studying the biological mechanisms of the new Corynebacterium species here described may shed light on their possible beneficial role for healthy FUM.

Urinary Microbiome of Reproductive-Age Asymptomatic European Women.

The knowledge of bacterial species diversity within the female urinary microbiome (FUM) is essential for understanding the role of the FUM in urinary tract health and disease. This study aimed to characterize the bacterial species diversity of the FUM of asymptomatic reproductive-age European women by combining extended culturomics and long-read sequencing of the near-full-length 16S rRNA gene. A total of 297 bacterial species (median of 53 species/sample) were identified, yet only 22% of the species were detected by both culture and sequencing methods. Recently recognized Gardnerella, Lactobacillus, and Limosilactobacillus species and 5 new putative Corynebacterium species were identified by culturomics, while anaerobic species (e.g., 11 Peptoniphilus spp.) were mostly detected by amplicon sequencing. Notably, there was not a single species common to all samples, although members of the genus Lactobacillus were detected in all. Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus mulieris were observed in high relative abundance in several samples, as well as other species (e.g., Streptococcus agalactiae, Fannyhessea vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while low-abundance members (e.g., Finegoldia magna) were often more prevalent. A moderate correlation (Mantel test; r = 0.5) between community structure types captured by culturomics and amplicon sequencing was observed, highlighting the benefit of combining both methodologies. This study provided a detailed FUM structure at the species level, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders. Moreover, the different capacity to characterize microbiome profiles of culturomic and amplicon sequencing is described, providing valuable insights for further urinary microbiome studies. IMPORTANCE The bacterial species diversity within the female urinary microbiome (FUM) has been insufficiently characterized. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported species belonging to the genera Lactobacillus, Limosilactobacillus, and Latilactobacillus and putative novel Corynebacterium species. Although some species were present in high relative abundance, low-abundance members were more prevalent. FUM classification into community structure types demonstrated high interindividual differences in urinary microbiome composition among asymptomatic women. We also report moderate correlation between culture-dependent and -independent derived data-highlighting drawbacks of each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of the FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

Virulence potential of the first Corynebacterium mycetoides strain isolated from human urine: a rare species of Corynebacterium.

For many years, the potential pathogenic of non-diphtheriae corynebacteria were underestimated. Nowadays, a growing number of Corynebacterium species are recognized as opportunistic agents of human infections, mainly in hospital settings. In addition, multidrug-resistant Corynebacterium isolates from clinical specimens, have been reported and the role of Corynebacterium spp. in urinary tract infections (UTIs) has been highlighted. Several studies have reported Corynebacterium species as the agent of UTIs especially in patients with risk factors. Thus, the present work aimed to report the first isolation of Corynebacterium mycetoides from human urine and an initial study on its virulence properties. The isolate, initially characterized by phenotypical tests as a multidrug-resistant Corynebacterium sp., was recovered from the urine of a female transplant patient. Mass spectrometry and 16S rRNA and rpoB genes sequencing identified the isolate as C. mycetoides. The isolate was found able to adhere to and survive into epithelial cells (Vero cells), and its pathogenic potential was confirmed when tested against Caenorhabditis elegans nematode. The results obtained suggest that C. mycetoides is a potential pathogen for the urinary tract in humans and for a better understanding of the multifactorial mechanisms of virulence, studies about this species should be continued.

Insights of OxyR role in mechanisms of host-pathogen interaction of Corynebacterium diphtheriae.

Corynebacterium diphtheriae, the leading causing agent of diphtheria, has been increasingly related to invasive diseases, including sepsis, endocarditis, pneumonia, and osteomyelitis. Oxidative stress defense is required not only for successful growth and survival under environmental conditions but also in the regulation of virulence mechanisms of human pathogenic species, by promoting mucosal colonization, survival, dissemination, and defense against the innate immune system. OxyR, functioning as a negative and/or positive transcriptional regulator, has been included among the major bacterial coordinators of antioxidant response. OxyR was first reported as a repressor of catalase expression in C. diphtheriae. However, the involvement of OxyR in C. diphtheriae pathogenesis remains unclear. Accordingly, this work aimed to investigate the role of OxyR in mechanisms of host-pathogen interaction of C. diphtheriae through the disruption of the OxyR of the diphtheria toxin (DT)-producing C. diphtheriae CDC-E8392 strain. The effects of OxyR gene disruption were analyzed through interaction assays with human epithelial cell lines (HEp-2 and pneumocytes A549) and by the induction of experimental infections in Caenorhabditis elegans nematodes and Swiss Webster mice. The OxyR disruption exerted influence on NO production and mechanism accountable for the expression of the aggregative-adherence pattern (AA) expressed by CDC-E8392 strain on human epithelial HEp-2 cells. Moreover, invasive potential and intracytoplasmic survival within HEp-2 cells, as well as the arthritogenic potential in mice, were found affected by the OxyR disruption. In conclusion, data suggest that OxyR is implicated in mechanisms of host-pathogen interaction of C. diphtheriae.