Antibiotic resistance in Escherichia coli in the female microbiota. Phylogenetic differences within the Escherichia coli.

Urinary tract infections (UTIs) are the most common infection in women. While UTIs are most frequently caused by E. coli, it can also reside within the urinary tract as a commensal member of the urinary microbiota. Prior genomic analysis of E. coli strains associated with UTIs and commensal strains have been conducted, looking at virulence factors and antibiotic resistance distinguishing the two. UTIs are commonly treated with an antibiotic. While E. coli strains can encode for antibiotic-resistance genes naturally, they can also acquire resistance because of prior antibiotic treatment. Dueto the community-acquired antibiotic resistance, there are less and less treatment options for UTIs. Recently we isolated and sequenced the genomes of 66 E. coli isolates from the bladder microbiota of women with UTIs, with urinary urgency incontinence, overactive bladder, and without LUTs. The efficacy of five commonly prescribed antibiotics on the growth of these strains was tested. Despite the presence of coding regions associated with antibiotic resistance, we found that UTI+ and UTI- strains exhibit similar sensitivities to these drugs. We also found that there is no difference within the UTI+ and UTI- strains in their placement in a phylogenetic tree based on their amino acid sequence

Exploring the Role of Lactobacillus Jensenii and Lactobacillus Mulieris in the Urogenital Tract

Lactobacillus is a predominant species of the urogenital tract and it has been found in females with and without lower urinary tract symptoms. The Lactobacillus genus is thought to be beneficial to the host due to its capability to produce hydrogen peroxide, secondary metabolites, and lactic acid. L. jensenii is an organism frequently isolated from the urogenital tract. In March 2020, a new sister taxon of L. jensenii was described, L. mulieris. Shortly thereafter we produced a genomic analysis of all publicly available genomes (n = 43) which reclassified some L. jensenii as L. mulieris. This motivated the in-depth study of the difference between L. jensenii and L. mulieris presented here which included expanding our collection of representatives of these two species to 61 strains. Genome analysis was conducted including the examination of their core genome as well as secondary metabolites, prophages and virulence factors. Complementing this genetic analysis, urinary strains of both species were phenotyped for urinary tract relevant characteristics, including sugar metabolism, pH and hydrogen peroxide production. Lastly, in an effort to ascertain the prevalence of these two species in the urinary tract, we found that the 16S rRNA gene sequence was insufficient, prompting our design of new gene markers that could specifically detect L. jensenii and L. mulieris while also being able to distinguish between the two. Using one of these gene markers, I assayed 190 urine samples and found that none of them contained either species. This led me to examine 233 urinary metagenomes for evidence of these gene markers, finding L. jensenii in only six samples and L. mulieris in two samples. These results suggest that L. jensenii and L. mulieris are not as abundant in the urinary microbiota as previously thought

Draft Genome Assemblies of 4 Lactobacillus jensenii and 3 Lactobacillus mulieris Strains from the Urinary Tract

Lactobacilli are dominant members of the healthy female bladder microbiota. Here, we report the draft genome sequences of 4 Lactobacillus jensenii and 3 Lactobacillus mulieris strains isolated from catheterized urine samples

Genomic insights into Lactobacillus gasseri and Lactobacillus paragasseri

Background. Antimicrobial and antifungal species are essential members of the healthy human microbiota. Several different species of lactobacilli that naturally inhabit the human body have been explored for their probiotic capabilities including strains of the species Lactobacillus gasseri. However, L. gasseri (identified by 16S rRNA gene sequencing) has been associated with urogenital symptoms. Recently a new sister taxon of L. gasseri was described: L. paragasseri. L. paragasseri is also posited to have probiotic qualities. Methods. Here, we present a genomic investigation of all (nD79) publicly available genome assemblies for both species. These strains include isolates from the vaginal tract, gastrointestinal tract, urinary tract, oral cavity, wounds, and lungs. Results. The two species cannot be distinguished from short-read sequencing of the 16S rRNA as the full-length gene sequences differ only by two nucleotides. Based upon average nucleotide identity (ANI), we identified 20 strains deposited as L. gasseri that are in fact representatives of L. paragasseri. Investigation of the genic content of the strains of these two species suggests recent divergence and/or frequent gene exchange between the two species. The genomes frequently harbored intact prophage sequences, including prophages identified in strains of both species. To further explore the antimicrobial potential associated with both species, genome assemblies were examined for biosynthetic gene clusters. Gassericin T and S were identified in 46 of the genome assemblies, with all L. paragasseri strains including one or both bacteriocins. This suggests that the properties once ascribed to L. gasseri may better represent the L. paragasseri species

Exploring the genotypic and phenotypic differences distinguishing Lactobacillus jensenii and Lactobacillus mulieris

Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii are dominant species of the urogenital microbiota. Prior studies suggest that these Lactobacillus species play a significant role in the urobiome of healthy females. In our prior genomic analysis of all publicly available L. jensenii and Lactobacillus mulieris genomes at the time (n = 43), we identified genes unique to these two closely related species. This motivated our further exploration here into their genotypic differences as well as into their phenotypic differences. First, we expanded genome sequence representatives of both species to 61 strains, including publicly available strains and nine new strains sequenced here. Genomic analyses conducted include phylogenetics of the core genome as well as biosynthetic gene cluster analysis and metabolic pathway analyses. Urinary strains of both species were assayed for their ability to utilize four simple carbohydrates. We found that L. jensenii strains can efficiently catabolize maltose, trehalose, and glucose, but not ribose, and L. mulieris strains can utilize maltose and glucose, but not trehalose and ribose. Metabolic pathway analysis clearly shows the lack of treB within L. mulieris strains, indicative of its inability to catabolize external sources of trehalose. While genotypic and phenotypic observations provide insight into the differences between these two species, we did not find any association with urinary symptom status. Through this genomic and phenotypic investigation, we identify markers that can be leveraged to clearly distinguish these two species in investigations of the female urogenital microbiota

Comparative Genomic Study of Lactobacillus jensenii and the Newly Defined Lactobacillus mulieris Species Identifies Species-Specific Functionality

Lactobacilli are dominant members of the “healthy” female urogenital microbiota. One of these species, Lactobacillus jensenii, is routinely identified in the urinary microbiota of women both with and without urinary tract symptoms. In March 2020, the new bacterial species Lactobacillus mulieris was introduced, and phylogenetic and average nucleotide identity analysis identified eight L. jensenii strains that should be classified as members of the L. mulieris species. This prompted our phylogenomic study of all publicly available L. jensenii and L. mulieris genome sequences. While there is little variation in the 16S rRNA gene sequences, the core genome shows a clear distinction between genomes of the two species. We find eight additional strains of the species L. mulieris among these genomes. Furthermore, one strain, currently classified as L. mulieris UMB7784, is distinct from both L. jensenii and L. mulieris strains. As part of our comparative genomic study, we also investigated the genetic content that distinguishes these two species. Unique to the L. jensenii genomes are several genes related to catabolism of disaccharides. In contrast, L. mulieris genomes encode several cell surface and secreted proteins that are not found within the L. jensenii genomes. These L. jensenii-specific and L. mulieris-specific loci provide insight into phenotypic differences of these two species

Comparative Genomic Study of Streptococcus anginosus Reveals Distinct Group of Urinary Strains

Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of healthy asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota. IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups

Vagococcus fluvialis isolation and sequencing from urine of healthy cattle

While the gram-positive bacterium Vagococcus fluvialis has been isolated from the environment as well as fish, birds, and mammals, very little is known about the species. V. fluvialis is believed to be a probiotic in fishes. However, within mammals, it is more frequently isolated from infectious tissue, including on rare occasions human and livestock lesions. Prior to the study described here, V. fluvialis had never been found in healthy bovine animals. Here, we present the complete genomes of V. fluvialis UFMG-H6, UFMG-H6B, and UFMG-H7, novel strains isolated from urine samples from healthy bovine females. These are the first genomes of mammalian isolates and the first description of V. fluvialis from urine. The genomes did not encode for any known virulence genes, suggesting that they may be commensal members of the urine microbiota

Coliphages of the human urinary microbiota

Due to its frequent association with urinary tract infections (UTIs), Escherichia coli is the best characterized constituent of the urinary microbiota (urobiome). However, uropathogenic E. coli is just one member of the urobiome. In addition to bacterial constituents, the urobiome of both healthy and symptomatic individuals is home to a diverse population of bacterial viruses (bacteriophages). A prior investigation found that most bacterial species in the urobiome are lysogens, harboring one or more phages integrated into their genome (prophages). Many of these prophages are temperate phages, capable of entering the lytic cycle and thus lysing their bacterial host. This transition from the lysogenic to lytic life cycle can impact the bacterial diversity of the urobiome. While many phages that infect E. coli (coliphages) have been studied for decades in the laboratory setting, the coliphages within the urobiome have yet to be cataloged. Here, we investigated the diversity of urinary coliphages by first identifying prophages in all publicly available urinary E. coli genomes. We detected 3,038 intact prophage sequences, representative of 1,542 unique phages. These phages include both novel species as well as species also found within the gut microbiota. Ten temperate phages were isolated from urinary E. coli strains included in our analysis, and we assessed their ability to infect and lyse urinary E. coli strains. We also included in these host range assays other urinary coliphages and laboratory coliphages. The temperate phages and other urinary coliphages were successful in lysing urinary E. coli strains. We also observed that coliphages from non-urinary sources were most efficient in killing urinary E. coli strains. The two phages, T2 and N4, were capable of lysing 83.5% (n = 86) of strains isolated from females with UTI symptoms. In conclusion, our study finds a diverse community of coliphages in the urobiome, many of which are predicted to be temperate phages, ten of which were confirmed here. Their ability to infect and lyse urinary E. coli strains suggests that urinary coliphages may play a role in modulating the E. coli strain diversity of the urobiome

Examination of staphylococcus aureus prophages circulating in Egypt

Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Re-cently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton–Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt