Genome sequences and annotation of two urinary isolates of E. coli
© The Author(s). 2016
Received: 13 May 2016
Accepted: 4 October 2016
Published: 12 October 2016
The genus Escherichia includes pathogens and commensals. Bladder infections (cystitis) result most often from colonization of the bladder by uropathogenic E. coli strains. In contrast, a poorly defined condition called asymptomatic bacteriuria results from colonization of the bladder with E. coli strains without symptoms. As part of an on-going attempt to identify and characterize the newly discovered female urinary microbiota, we report the genome sequences and annotation of two urinary isolates of E. coli: one (E78) was isolated from a female patient who self-reported cystitis; the other (E75) was isolated from a female patient who reported that she did not have symptoms of cystitis. Whereas strain E75 is most closely related to an avian extraintestinal pathogen, strain E78 is a member of a clade that includes extraintestinal strains often found in the human bladder. Both genomes are uncommonly rich in prophages.
Clinicians typically equate the presence of bacteria in urine with infection, or, less commonly, an ill-defined phenomenon termed “asymptomatic bacteriuria.” These and other existing concepts are based on the long-held “sterile urine” paradigm. Recently, however, bacterial communities (microbiota) have been discovered in the female bladder [1–9]. Thus, the “sterile urine” paradigm is no longer valid.
In an effort to provide a comprehensive view of the newly discovered female urinary microbiota, we have established an Enhanced Quantitative Urine Culture protocol. This enhanced culture protocol isolates bacteria from 75 to 90 % of urine samples deemed ‘no growth’ by the standard clinical microbiology urine culture method [4, 7, 10]. We have begun to the sequence and annotate the genomes of these isolated bacteria.
Here, we report the full genome sequences and annotations of two of those bacteria, Escherichia coli strains E75 and E78 isolated from female patients pursuing urogynecologic clinical care. Strain E75 was isolated from a patient who thought that she did not have a urinary tract infection, while E78 was isolated from a patient who thought that she did. The strains were sub-cultured to purity and then identified as E. coli by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry . Strain E75 is most closely related to APEC O1, an avian extraintestinal pathogen. In contrast, strain E78 is a member of a clade that includes extraintestinal strains often associated with the human bladder, including uropathogenic strains UTI89 and J89 and asymptomatic bacteriuric strain ABU83972. Both genomes are uncommonly rich in prophages.
Classification and features
Escherichia coli is a non-sporulating, Gram-negative, rod shaped bacterium. It is a facultative anaerobe found commonly in the environment and the lower intestines of mammals and other endotherms. Extra-intestinal strains can colonize other organs, including the urinary bladder. Most E. coli strains are harmless constituents of the normal microbiota, but others cause disease. For example, uropathogenic E. coli is the major case of urinary tract infections in humans; other E. coli strains colonize the bladder without causing symptoms, a condition called asymptomatic bacteriuria.
Classification and general features of E. coli strains E75 and E78
Species Escherichia coli
Strain: E75 and E78
pH range; Optimum
Not determined, strains grown in complex medium
Human female bladder
0.5 % (w/v)
Suspected pathogen (E78)
Maywood, IL USA
E75 (9/14/2014); E78 (9/25/2014)
E. coli strains E75 and E78 were isolated from patients who sought clinical care at Loyola University Medical Center’s Female Pelvic Medicine and Reconstructive Surgery center in September 2014. Patients were asked the question: Do you feel that you have a urinary tract infection? E75 was isolated from a patient who answered ‘no,’ whereas E78 was isolated from a patient who answered ‘yes.’ Both patients were white, post-menopausal women seeking care for Pelvic Organ Prolapse. Neither patient was taking antibiotics; both were using daily vaginal estrogen supplement. The UTI Symptoms Assessment Questionnaire was used to characterize the degree of severity and bother of the patients’ symptoms . Both E. coli strains were identified at >100,000 colony forming units per milliliter, using an Expanded Spectrum version of the Enhanced Quantitative Urine Culture protocol . After they were sub-cultured to purity, Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry was used to confidently identify them as E. coli . For E75, the identification score was 2.530; for E78, the score was 2.265. No other microbes were detected in the urine sample containing strain E75. In the urine sample containing strain E78, Alloscardovia omnicolens (10 colony forming units per milliliter) and Lactobacillus rhamnosus (10 colony forming units per milliliter) were also detected.
Genome sequencing information
Genome project history
High quality draft
High quality draft
Paired-end library of 150 bp
Paired-end library of 150 bp
Gene calling method
GenBank Date of Release
May 9, 2016
May 9, 2016
Source Material Identifier
Growth conditions and genomic DNA preparation
E. coli strains E75 and E78 were isolated from transurethral catheterized urine specimens of adult women with urinary symptoms  using a Expanded Spectrum version of the previously described Enhanced Quantitative Urine Culture protocol . Three urine volumes (1 μL, 10 μL, and 100 μL) of each urine sample was spread quantitatively (i.e., pinwheel streak) onto t5% sheep blood (BD BBL™ Prepared Plated Media, Cockeysville, MD), Chocolate, and Colistin Naladixic Acid agars (BD BBL™ Prepared Plated Media) and incubated in 5 % CO2 at 35 °C for 48 h; 5 % sheep blood and MacConkey (BD BBL™ Prepared Plated Media) agars incubated aerobically at 35 °C for 48 h; two CDC Anaerobic 5 % sheep blood agars (BD BBL™ Prepared Plated Media) incubated in either Microaerophilic Campy gas mixture (5 % O2, 10 % CO2, 85 % N), or anaerobically at 35 °C for 48 h. All agars were documented for growth (i.e., for morphologies and colony forming units per milliliter) at 24 and 48 h. Each distinct colony morphology was sub-cultured at 48 h to obtain pure culture for microbial identification.
Microbial identification was determined using a Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometer (Bruker Daltonics, Billerica, MA) as described . Pure cultures were stored at -80 °C in a 2 ml CryoSaver Brucella Broth with 10 % Glycerol, no beads, Cryovial, for preservation (Hardy Diagnostics). For genome extraction and sequencing, the preserved pure culture isolates were grown on 5 % sheep blood agar under aerobic conditions at 35 °C for 24 h.
Genomic DNA extraction was performed using a phenol-chloroform extraction protocol. Briefly, cells were resuspended in 0.5 mL DNA Extraction Buffer (20 mM Tris-Cl, 2 mM EDTA, 1.2 % Triton X-100, pH 8) followed by addition of 50uL Lysozyme (20 mg/mL), 30uL Mutanolysin, and 5uL RNase (10 mg/mL). After a 1 h incubation at 37 °C, 80uL 10 % SDS, and 20uL Proteinase K were added followed by a 2 h incubation at 55 °C. 210uL of 6 M NaCl and 700uL phenol-chloroform were then added. After a 30-min incubation with rotation, the solutions were centrifuged at 13,500 RPM for 10 min, and the aqueous phase was extracted. An equivalent volume of Isopropanol was then added, and solution was centrifuged at 13,500 RPM for 10 min after a 10-min incubation. The supernatant was decanted and the DNA pellet was precipitated using 600uL 70 % Ethanol. Following ethanol evaporation, the DNA pellet was resuspended in Tris-EDTA and stored at -20 °C.
Genome sequencing and assembly
DNA samples were diluted in water to a concentration of 0.2 ng/ul as measured by a fluorometric-based method (Life Technologies, Carlsbad, CA) and 5 ul was used to obtain a total of 1 ng of input DNA. Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to manufacturer’s instructions. The isolates were barcoded, pooled and each isolate was sequenced twice, on two separate runs, using the Illumina MiSeq platform and the MiSeq Reagent Kit v2 (300-cycles) to produce 150 bp paired-end reads. Sequencing reads were parsed into individual folders according to the respective barcodes.
Summary of genomes: two chromosomes and two plasmids
Genes were identified using GLIMMER using the g3-from-scratch.csh script included in the package  The predicted CDSs were translated using the transeq script within the EMBOSS suite . rRNA genes were identified by RNAmmer  using the parameter set to identify bacterial rRNA sequences. The program tRNA-Scan  identified tRNA sequences, using the parameter for bacterial tRNAs. Trans-membrane proteins were identified using TMHMM with standard parameters . SignalP  predicted signal peptides. All CDSs were queried (blastp) locally against the COG sequence dataset () and assigned based upon their sequence homologies. CRISPR elements were detected through CRISPR-db . Genes with Pfam domains were ascertained via searches of the Pfam database (E-value threshold 1.0) .
% of Totala
% of Totala
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights from the genome sequence
Although E75 was isolated from a woman who reported that she did not have symptoms of cystitis, its genome encodes proteins associated with E. coli pathogenesis, including the P pilus, RTX toxin, and α-fimbriae. These genes were not found in E78. While the E75 strain did not include plasmid sequences, genome sequencing of the E78 isolate contained two. Plasmid pE78.2 was nearly identical (one mismatch) to the E. coli plasmid pVR50G, collected from urine obtained from an individual with asymptomatic bacteriuria .
Predicted sequences of phage origin and putative origin
Number of predicted phage CDSs
Exhibit no sequence homology to GenBank
Species with most hits (# hits)
Enterobacteria phage lambda (19)
Bacteriophage P4 (10)
The genome of E75, isolated from a woman who reported no symptoms of cystitis, is more closely related to the avian extraintestinal pathogen APEC 01. The genome of E75, isolated from a woman who reported cystitis symptoms, resides in a clade populated by human extra-intestinal strains that are either uropathogenic or asymptomatic bacteriuric. Both genomes contain an unusually large number of prophage sequences.
We would like to acknowledge Female Pelvic Medicine & Reconstructive Surgery at Loyola University Medical Center for their help in the patient recruitment and sample collection that led to isolation of these strains, Dr. Doerte Lehmann for aiding in the TEM sample preparation protocol, and Linda Fox from the Core Imaging Facility at Loyola University Chicago for obtaining the TEM images. We also acknowledge Gina Kuffel and Dr. Michael Zilliox for sequencing the genomes. AJW and CP are supported by Loyola University Chicago’s Multidisciplinary Research Award. This work was also partially funded by the NSF (1149387 to CP) and the NIH (R21DK097435-01A1 to AJW).
TKP conceived the project, isolated the bacteria, identified them by MALDI-TOF, and prepared them for sequencing. AM, KM, LK, and CP analysed the sequence data. EEH attained the TEM images. AJW conceived the project and oversaw its progress. AJW and CP wrote the manuscript. All authors read and edited the manuscript. All authors read and approved the final manuscript.
The authors have no competing interests to report.
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