Finished Genome Sequence of the Highly Multidrug-Resistant Human Urine Isolate Citrobacter freundii Strain SL151

Citrobacter freundii is a Gram-negative opportunistic pathogen that is increasingly being recognized as a causative agent of hospital-acquired urinary tract infections and an important reservoir of antimicrobial resistance determinants. In this report, we describe the finished genome sequence of C. freundii strain SL151, a highly multidrug-resistant human urine isolate.

pathogen and is known to cause a variety of ailments (e.g., urinary tract infections [UTIs], wound infections, gastrointestinal infections, septicemia, meningitis), especially in immunocompromised patients and in hospital settings (1)(2)(3)(4)(5). This emergence has coincided with the finding that C. freundii is often resistant to multiple classes of antibiotics, suggesting that both clinical and environmental strains may be important reservoirs of antimicrobial resistance determinants (ARDs) (6)(7)(8)(9)(10). For example, a recent survey of outpatients in Bo, Sierra Leone, revealed that a surprisingly high number of C. freundii were isolated from the urine of individuals with UTI symptoms and that all of these isolates were highly multidrug-resistant (MDR) (11). To determine the underlying genetics responsible for these observed MDR phenotypes, we sequenced the genome of a representative isolate from this collection, C. freundii strain SL151, using the Pacific Biosciences RS II sequencing platform (DNA Link USA, Inc., San Diego, CA, USA).
Genomic DNA was extracted using the Gentra Puregene yeast/ bacteria Kit (Qiagen, Valencia, CA, USA) and used to prepare a 20-kb insert library that was sequenced using a single-molecule real-time (SMRT) sequencing cell and P6-C4 chemistry. This resulted in 7,148 filtered and preassembled sequence reads with a mean length of 17,444 bp and 23ϫ genome coverage. Assembly and consensus polishing (via SMRT Analysis version 2.3.0 and HGAP.2) yielded one circular chromosome (5,073,255 bp, 51.7% GC) and two circular plasmids (210,673 bp, 54.7% GC, and 154,967 bp, 53.7% GC) with a finished genome size of 5,438,895 bp. Gene prediction and annotation were performed using GeneMarkSϩ and the NCBI Prokaryotic Genome Annotation Pipeline, respectively, and identified 5,537 coding sequences, of which 1,127 were predicted to encode hypothetical proteins.
A standard BLAST query of the Comprehensive Antibiotic Resistance Database (CARD) (12) using the SL151 genome sequence resulted in the identification of 97 resistance-associated genes, of which 36 demonstrated Ն95% nucleotide identity to the CARD reference sequences and provided the underlying genotype for every observed resistance phenotype. ARDs found on the chro-mosome include the intrinsic C. freundii AmpC cephalosporinase encoded by the bla CMY-79 gene, a truncated copy of a qnrB gene, and a number of multidrug efflux pump-encoding genes. The larger plasmid contained the aac(3)-III, bla TEM-1 , and bla CTX-M-15 genes in a Tn2-like structure identical to Klebsiella pneumoniae pENVA (13), as well as the qnrS1, sul2, catA1, strA, strB, and aadA16 genes. Finally, the smaller plasmid was found to contain an~11-kb-long, ARD-rich, composite class 1 integron harboring aac(6=)-Ib-cr, arr-3, dfrA27, aadA16, and qnrB6 gene cassettes. This composite element is nearly identical to structures found in K. pneumoniae and K. oxytoca plasmids (14,15). Interestingly, a mutated duplication of this region was found adjacent to the fully functional copy. Also detected was an IS26-flanked composite transposon containing the catA2 and tetD genes that is identical to a locus from the Salmonella sp. plasmid pTC67. Overall, this genome highlights the mosaic plasmid-mediated accumulation of ARDs in C. freundii, particularly those ARDs conferring resistance to aminoglycoside, fluoroquinolone, and sulfonamide compounds.
Accession number(s). This whole-genome project has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP016952, CP017058, and CP017059.

ACKNOWLEDGMENTS
We thank Jon Choi and Gun Eui Lee (DNA Link USA, Inc.) for their sequencing and assembly support. This work was supported by the Office of Naval Research via U.S. Naval Research Laboratory core funds. The opinions and assertions contained herein are those of the authors and are not to be construed as those of the U.S. Navy, military service at large, or the U.S. Government.

FUNDING INFORMATION
This work, including the efforts of Gary J. Vora, was funded by DOD | United States Navy | Office of Naval Research (ONR) (69-4888-06).