mEnrich-seq: methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome.

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that sequences specific bacterial taxa of interest, but not most background taxa. We developed mEnrich-seq (in which 'm' stands for methylation and seq for sequencing) for enriching taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self versus nonself differentiation by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes, individually or in combination, to deplete host and background taxa while enriching targeted taxa. This idea is integrated with library preparation procedures and applied in several applications to enrich (up to 117-fold) pathogenic or beneficial bacteria from human urine and fecal samples, including species that are hard to culture or of low abundance. We assessed 4,601 bacterial strains with mapped methylomes so far and showed broad applicability of mEnrich-seq. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest.

Gardnerella pickettii sp. nov. (formerly Gardnerella genomic species 3) and Gardnerella greenwoodii sp. nov. (formerly Gardnerella genomic species 8) isolated from female urinary microbiome.

During an ongoing female urinary microbiome research study, strains c17Ua_112T and c31Ua_26T isolated from urine samples of a patient diagnosed with overactive bladder and a healthy postmenopausal woman, respectively, could not be allocated to any Gardnerella species with valid names. In this work, we aimed to characterize these strains. The 16S rRNA gene sequences confirmed that these strains are members of the genus Gardnerella. Phylogenetic analysis based on cpn60 strongly supported two clades, one encompassing c17Ua_112T and nine other strains from the public database, and the other including c31Ua_26T and three other strains, which were distinct from currently recognized species of the genus Gardnerella. Likewise, the phylogenomic tree also showed that strains c17Ua_112T and c31Ua_26T formed independent and robust clusters. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between c17Ua_112T and c31Ua_26T were 79.27 and 27.4 %, respectively. Strain c17Ua_112T showed the highest ANI (94.8 %) and dDDH values (59.8 %) with Gardnerella piotii UGent 18.01T, and strain c31Ua_26T revealed highest ANI (84.2 %) and dDDH (29.1 %) values with Gardnerella swidsinskii GS 9838-1T. Based on the data presented here, the two strains c17Ua_112T and c31Ua_26T represent two novel species of the genus Gardnerella, for which the names Gardnerella pickettii (c17Ua_112T=DSM 113414T=CCP 71T) and Gardnerella greenwoodii (c31Ua_26T=DSM 113415T=CCP 72T) are proposed.

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.

Phylogenomic analysis of a highly virulent Escherichia coli ST83 lineage with potential animal-human transmission.

The presence of specific virulence features conditions severe forms of urinary tract disease, but the frequency and distribution of these highly virulent extraintestinal pathogenic Escherichia coli strains in animals and humans is unclear. We used whole genome sequencing, comparative genomics, histological and clinical data to characterize the genetic basis for pathogenesis and origin of E. coli Ec_151217, a strain (B2, ST83, O83:H5:K5) that caused an extremely aggressive upper urinary tract infection (UTI) in a cat. We show that Ec_151217 and 52% of other highly related ST83 genomes (O6 and O83) identified from different animal species and human infections carry two copies of the hemolysin A operon, though this duplication is infrequent (~1%) among closed ExPEC genomes from multiple sources. Our data enlarges the list of E. coli genetic backgrounds carrying hlyA operon duplication which is potentially involved in severity of UTI, and demonstrates that it seems to occur infrequently amongst ExPEC. Its identification in E. coli lineages (diverse ST83 serotypes) of potential animal-human transmission is of concern and anticipates the need to screen larger collections.

Long-term stability of the urogenital microbiota of asymptomatic European women.

BACKGROUND: To date, information on healthy female urinary microbiota is available mostly at genus level and at one time point. However, profound species-level characterization of healthy urinary microbiome and its stability over time are essential for further correct interpretation of its role in healthy urogenital tract. In this study, we investigated female urogenital microbiome (FUM) at two timepoints (within 2.5-year interval) in young asymptomatic European women. We used culturomics with accurate isolates' identification (MALDI-TOF MS and gene markers sequencing) to understand species stability within healthy FUM. RESULTS: Extended culturomics of voided midstream urine sample pairs revealed a mean Shannon diversity index of 1.25 and mean of 19 species/sample (range 5-39 species; total of 115 species; 1830 isolates). High overall species variability between individuals was captured by beta diversity and a variety of community structure types, with the largest cluster characterized by Lactobacillus crispatus, often in combination with Gardnerella vaginalis or Gardnerella genomospecies 3. Significant FUM composition differences, related to Finegoldia magna and Streptococcus anginosus, according to smoking status were found. A high species variability within individuals (Shannon index SD > 0.5 in 7 out of 10 sample pairs) with a mean of 29% of shared species (range 9.1-41.7%) was observed. Moreover, 4 out of 10 sample pairs clustered in the same community structure type. The stable FUM sample pairs presented high abundance of Lactobacillus crispatus, Streptococcus agalactiae or Lactobacillus paragasseri and Bifidobacterium spp.. Moreover, Gardnerella vaginalis, Gardnerella genomospecies 3 or Gardnerella swidsinskii were often maintained within individuals in high abundance. CONCLUSIONS: Shift in species composition at two distant timepoints was frequently observed among urogenital microbiome of European asymptomatic women. This suggests possible interchange of particular species in healthy FUM and the existence of multiple health-associated FUM compositions in certain individuals. Additionally, we provided additional evidence on resilience of particular bacterial communities and identified certain species more prone to persist in urogenital tract. This study revealed important details on the FUM composition complexity relevant for studies aiming to understand microbiota role in the urogenital tract health and for identification of eubiotic and dysbiotic FUM.

MicroMundo@UPorto: an experimental microbiology project fostering student's antimicrobial resistance awareness and personal and social development.

Antimicrobial resistance (AMR) is a global societal challenge requiring the contribution of professionals along with general community citizens for their containment. Portugal is one of the European countries where a lack of knowledge on the correct use of antimicrobials and AMR problematic is preeminent. Moreover, youth demotivation to pursue science careers is emerging. To address these problems an innovative experimental service-learning pedagogical strategy, MicroMundo@UPorto, was implemented in Portugal during 2018 through University of Porto as a partner of the global Citizen Science project 'Tiny Earth' responding to the AMR crisis. In this first edition of MicroMundo@UPorto, university students (n = 41; Pharmaceutical Sciences and Nutrition Sciences) organized in eight teams tutored by university professors/researchers (n = 13) on Microbiology and AMR theoretical and practical aspects as well on communication skills to enable their guidance of younger school students (n = 140/3 schools) in experiments to discover antimicrobial-producing microorganisms while exploring the soil microbial diversity. Post-survey-based evaluation revealed that this project allowed university students to acquire diverse personal, social and scientific skills while increasing AMR awareness, in the One-Health perspective, and interest for science in school students. This University to Society approach can be successfully extended across Portugal and for education in Microbiology in general, with benefits for the future generations contributing to socially responsible and scientifically-literate citizens.

The Darkest Place Is under the Candlestick-Healthy Urogenital Tract as a Source of Worldwide Disseminated Extraintestinal Pathogenic Escherichia coli Lineages.

Since the discovery of the urinary microbiome, including the identification of Escherichia coli in healthy hosts, its involvement in UTI development has been a subject of high interest. We explored the population diversity and antimicrobial resistance of E. coli (n = 22) in the urogenital microbiome of ten asymptomatic women (representing 50% of the sample tested). We evaluated their genomic relationship with extraintestinal pathogenic E. coli (ExPEC) strains from healthy and diseased hosts, including the ST131 lineage. E. coli prevalence was higher in vaginal samples than in urine samples, and occasionally different lineages were observed in the same individual. Furthermore, B2 was the most frequent phylogenetic group, with the most strains classified as ExPEC. Resistance to antibiotics of therapeutic relevance (e.g., amoxicillin-clavulanate conferred by blaTEM-30) was observed in ExPEC widespread lineages sequence types (ST) 127, ST131, and ST73 and ST95 clonal complexes. Phylogenomics of ST131 and other ExPEC lineages revealed close relatedness with strains from gastrointestinal tract and diseased host. These findings demonstrate that healthy urogenital microbiome is a source of potentially pathogenic and antibiotic resistant E. coli strains, including those causing UTI, e.g., ST131. Importantly, diverse E. coli lineages can be observed per individual and urogenital sample type which is relevant for future studies screening for this uropathogen.

The success of particular Acinetobacter baumannii clones: accumulating resistance and virulence inside a sugary shield.

BACKGROUND: In Portugal, carbapenem-resistant Acinetobacter baumannii (CRAB) has been associated with ST98, ST103 and ST208 (Oxford Scheme, Oxf) and a clone has usually been associated with a particular period of time. These clonal shifts were primarily explained by an increased antimicrobial resistance profile. Here we explore genomic and biochemical differences among these and more recent clones, which could further explain the diversity and evolution of this species. METHODS: A total of 116 CRAB isolates (2010-15), together with representatives of a previously described CRAB collection (4 isolates, 2001-06) were characterized by attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) and MLST. Representatives of different FTIR-ATR/MLST clusters were selected for WGS (n = 13), which allowed the in silico extraction of resistance and virulence genes, capsule locus and SNP analysis. RESULTS: A. baumannii clonal shifts of OXA-58-producing ST103Oxf (2001-04), OXA-40-producing ST98Oxf (2002-06), OXA-23-producing ST208Oxf (2006-10) and OXA-23-producing ST218Oxf (2010-15) were accompanied by an increase in AMR genes and virulence factors. FTIR-ATR clustering was congruent with sugar composition predicted from the capsular locus: a fucosamine cluster comprising ST98Oxf, ST103Oxf and a single ST218Oxf isolate; a pseudaminic acid cluster of ST208Oxf and ST1557Oxf isolates; and legionaminic acid, resembling the sialic acid from mammalian cells, in a cluster comprising ST218Oxf isolates. The whole-genome phylogenetic tree was congruent with MLST, with isolates presenting 5-28 938 SNPs. ST208Oxf and ST218Oxf presented ∼1900 SNPs while ST103Oxf and ST1557Oxf showed a greater number of SNPs (∼28 000). CONCLUSIONS: Clonal shifts of CRAB were promoted, in our country, by consecutive virulence and AMR gene pool enlargement, together with features increasing pathogen-host adaptation. Worldwide dominance of ST218Oxf is supported by the combination of high AMR and virulence levels.

The status of the species Lactobacillus fornicalis Dicks et al. 2000. Request for an opinion.

During a recent study on members of the genus Lactobacillus we realized that cultures of Lactobacillus fornicalis TV 1018T (=DSM 13171T=ATCC 700934T) are no longer available from the online catalogue of the German Collection of Microorganisms and Cell Cultures GmbH, being displayed as Lactobacillus plantarum at the American Type Culture Collection. Based on data currently available, the organism deposited as ATCC 700934T is a member of the species Lactobacillus plantarum subs. plantarum. Therefore, the type strain of Lactobacillus fornicalis cannot be included in any further scientific comparative study. This matter is referred to the Judicial Commission, asking for an opinion on the status of the species.

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus. Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii, respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus, for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

Limosilactobacillus urinaemulieris sp. nov. and Limosilactobacillus portuensis sp. nov. isolated from urine of healthy women.

Two Gram-stain-positive strains, c9Ua_26_MT and c11Ua_112_MT, were isolated from voided urine samples from two healthy women. Comparative 16S rRNA gene sequences demonstrated that these novel strains were members of the genus Limosilactobacillus. Phylogenetic analysis based on pheS gene sequences and core genomes showed that each strain formed a separated branch and are closest to Limosilactobacillus vaginalis DSM 5837T. The average nucleotide identity (ANI) and Genome-to-Genome Distance Calculator (GGDC) values between c9Ua_26_MT and the closest relative DSM 5837T were 90.7 and 42.9 %, respectively. The ANI and GGDC values between c11Ua_112_MT and the closest relative DSM 5837T were 91.2 and 45.0 %, and those among the strains were 92.9% and 51,0 %, respectively. The major fatty acids were C12 : 0 (40.2 %), C16 : 0 (26.7 %) and C18 : 1 ω9c (17.7 %) for strain c9Ua_26_MT, and C18 : 1 ω9c (38.0 %), C16 : 0 (33.3 %) and C12 : 0 (17.6 %) for strain c11Ua_112_MT. The genomic DNA G+C content of strains c9Ua_26_MT and c11Ua_112_MT was 39.9 and 39.7 mol%, respectively. On the basis of the data presented here, strains c9Ua_26_MT and c11Ua_112_MT represent two novel species of the genus Limosilactobacillus, for which the names Limosilactobacillus urinaemulieris sp. nov. (c9Ua_26_MT=CECT 30144T=LMG 31899T) and Limosilactobacillus portuensis sp. nov. (c11Ua_112_MT=CECT 30145T=LMG 31898T) are proposed.