MSACL 2016 EU Abstract

Use of the MALDI BioTyper System with MALDI-TOF MS for Rapid Identification of Microorganisms Causing Bacterial Urinary Tract Infection from Urine Samples

Kazuyuki Sogawa (Presenter)
Azabu University

Authorship: Kazuyuki Sogawa (1), Kaori Hayashi (1), Saori Abe (2), Tomohiro Sunagawa (2), Shigeko Tanigawa (2), Atsushi Kanaiwa (2), Mika Mishina (1), Toshifumi Watanabe (1), Hiroto Maeda (2), Katsunori Furuhata (1)
(1) Azabu University, Kanagawa, Japan (2) Maeda Veterinary Hospital, Hokkaido, Japan

Short Abstract

With the increasing number of cats maintained as pets, the opportunities to treat cats with lower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. We performed direct identification of bacteria in urine using pretreatment kits for the direct application of positive blood culture bottles to MALDI-TOF MS, aiming to improve the low identification rates of E. faecalis. The concordance rates of E. faecalis identification by the MALDI-TOF MS method employing the MALDI Sepsityper Kit for pretreatment were 83.3, showing that the rate was improved.

Long Abstract

Introduction: With the increasing number of cats maintained as pets, the opportunities to treat cats with lower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. Several studies have reported urinary tract infection of felines with LUTD accounting for 2–10% of cases [1, 2]. The causes of renal failure in young cats include renal hypoplasia, polycystic kidney, renal agenesis and urinary tract dysplasia. In middle-aged and elderly cats, urinary bacterial infection-induced cystitis, due to reduction of urine specific gravity accompanying glomerulonephritis and interstitial nephritis, is frequently observed [3, 4]. Furthermore, bacterial infections associated with renal failure caused by urinary tract stones formed by struvite (magnesium ammonium phosphate calculus) and calcium oxalate calculi and tumors develop regardless of the age, and progression to pyonephrosis occurs occasionally, increasing the importance of the identification of urinary bacteria and drug sensitivity tests. However, it can require several days to obtain results from a laboratory test center, which delays the initiation of appropriate treatment with antibiotics at clinical sites. We performed direct identification of bacteria in urine using pretreatment kits for the direct application of positive blood culture bottles to MALDI-TOF MS, aiming to improve the low identification rates of E. faecalis and 2-morphology colony types.

Methods: Urine samples collected from 39 cats with bacterial cystitis brought to Maeda Veterinary Hospital between August 10, 2015 and March 31, 2016 (Mix; 26 males and 13 females aged 0-19 years) were used in the study. Urine was collected by puncture of the urinary bladder with a 25-G needle through the skin after confirming the position of the urinary bladder in a supine position with ultrasound. Bacterial identification was performed by the conventional method using the MicroScan WalkAway system (Siemens Healthcare Diagnostics, IL, USA). Sample preparation of the urine was performed using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany) according to the manufacturer's instructions [5]. To identify the isolates, MALDI-TOF MS was performed on the AutoFlex® TOF/TOF mass spectrometer (Bruker Daltonics), in accordance with the manufacturer’s instructions, and with Flexcontrol™ software 3.0 (Bruker Daltonics) for the automatic acquisition of mass spectra in the linear positive mode within a range of 2 to 20 kDa.

Results: Of the 39 urine specimens, the growth of colonies was observed in 30 specimens: 24 specimens with single colony morphology, 6 specimens with two colony morphology. Nine specimens did not grow in culture, and MALDI-TOF MS did not identify any significant protein profile in any of these cases. Single colony morphology correctly identified 20 isolates (83.3%) using the MALDI Sepsityper Kit. In 4 cases in which identification was not possible, the bacterial count was 1.0 × 105 CFU/mL or lower, which was below the detection limit of sensitivity. In the specimens with two-colony morphology, the MALDI Sepsityper Kit identified only one species (Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae).

Conclusions: Urine culture (i.e., quantitative culture of urine specimens on solid medium followed by biochemical characterization of isolates), which is the gold standard for diagnosis, takes 24-72 hour until the results become available [6]. The MALDI-TOF MS method treats urine specimens with the MALDI Sepsityper Kit, and identifies bacterial species using MALDI BioTyper automation software within about 30 minutes. The MALDI-TOF MS method employing the MALDI Sepsityper Kit for pretreatment is a quick and reliable method for the identification of bacteria from infected urines, whose shortened analysis time enables an earlier and more accurate selection of antibiotics for feline LUTD treatment.


References & Acknowledgements:

References

[1] Gunn-Moore D (2003) Feline lower urinary tract disease. J Feline Med Surg. 5:133-138.

[2] Gerber B, Boretti FS, Kley S, Laluha P, Müller C, Sieber N, Unterer S, Wenger M, Flückiger M, Glaus T, Reusch CE (2005) Evaluation of clinical signs and causes of lower urinary tract disease in European cats. J Small Anim Pract. 46:571-577.

[3] Osborne CA, Kruger JM, Lulich JP, Polzin DJ, Lekcharoensuk C (2000) Feline lower urinary tract disease. In: Textbook of Veterinary Internal Medicine. 5th ed. Eds S. J. Ettinger and E. C. Feldman. W. B. Saunders, Philadelphia. pp 1710-1747.

[4] Osborne CA, Kruger JM, Lulich JP (1996) Feline lower urinary tract disorders. Definition of terms and concepts. Veterinary Clinics of North America: Small Animal Practice 26:169-179.

[5] Ferreira L, Sánchez-Juanes F, González-Avila M, Cembrero-Fuciños D, Herrero-Hernández A, González-Buitrago JM, Muñoz-Bellido JL (2010) Direct identification of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 48:2110-2115.

[6] Burd EM, Kehl KS (2011) A critical appraisal of the role of the clinical microbiology laboratory in the diagnosis of urinary tract infections. J Clin Microbiol. 49:S34-S38.


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