NOTE
Bacteriology

Use of the MALDI BioTyper system andrapid BACpro with MALDI-TOF MS for rapid
identification of microorganisms causing
bacterial urinary tract infection in felineurine samples
Hiroto MAEDA1), Kaori HAYASHI2), Takayuki ISHIGE3), Tomohiro SUNAGAWA1),
Shigeko TANIGAWA1), Mika MISHINA4), Toshifumi WATANABE4) andKazuyuki SOGAWA2)*
1)Maeda Veterinary Hospital, 1-4-8 Yamatemachi, Tomakomai, Hokkaido 053-0851, Japan2)Department of Biochemistry, School of Life and Environmental Science, Azabu University,

1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan

3)Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
4)Veterinary Teaching Hospital, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara,

Kanagawa 252-5201, Japan

J. Vet. Med. Sci.
80(10): 1490–1494, 2018
doi: 10.1292/jvms.18-0145
Received: 16 March 2018
Accepted: 30 July 2018
Published online in J-STAGE:
9 August 2018

ABSTRACT. With the increasing number of cats kept as pets, opportunities to treat cats withlower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. Urine
samples collected from 50 cats with bacterial cystitis brought to Maeda Veterinary Hospital
between August 10, 2015 and March 31, 2017 were used in the study. Sample preparation of
the urine was performed using a MALDI Sepsityper kit and rapid BACpro. To identify the isolates,
MALDI-TOF MS was performed on an AutoFlex TOF/TOF mass spectrometer. MALDI-TOF MS using
rapid BACpro for pretreatment was found to be a quick and reliable method for identification ofbacteria from infected urine, with a shortened analysis time enabling earlier and more accurate
selection of antibiotics for treatment of feline LUTD.
KEY WORDS: cat, lower urinary tract disease, matrix-assisted laser desorption ionization-time offlight mass spectrometry, urine

The increasing number of cats kept as pets has increased opportunities to treat cats with lower urinary tract disease (LUTD) inclinical veterinary medicine. Urinary tract infection accounts for 2–10% of cases of feline LUTD [6, 7]. The causes of renal failure
in young cats include renal hypoplasia, polycystic kidney, renal agenesis, and urinary tract dysplasia, whereas urinary bacterial
infection-induced cystitis due to reduction of urine specific gravity accompanying glomerulonephritis and interstitial nephritis is
more common in middle-aged and elderly cats [11, 12]. Bacterial infections associated with renal failure caused by urinary tractstones formed by struvite (magnesium ammonium phosphate) and calcium oxalate calculi and tumors develop regardless of age,and progression to pyonephrosis may occur. This increases the importance of identification of urinary bacteria and drug sensitivity
tests. However, several days may be required to obtain results from a laboratory test center, which delays initiation of appropriate
treatment with antibiotics at clinical sites.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used to generate protein
fingerprint signatures from whole bacterial cells [4]. Algorithms can then be used for rapid identification of bacteria by comparing
fingerprints to a database of reference spectra [4]. Anhalt and Fenselau performed the first study using MS to identify bacteria[1]. However, MALDI-TOF MS instruments for use under routine conditions have only recently become available. For urine
specimens with ≥105 CFU/ml, the Escherichia coli identification rate is high (97.6–100.0%), but that for Enterococcus faecalis islow (60.0–66.7%), showing variation of rates among bacterial species [5, 8]. The identification rate for two-morphology colony
types is also low (60%, 3/5) [5].
In this study, we performed direct identification of bacteria in urine using pretreatment kits for direct application of positive
blood culture bottles to MALDI-TOF MS, with the aim of improving the low identification rates for E. faecalis and two*Correspondence to: Sogawa, K.: sogawa@azabu-u.ac.jp©2018 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/)

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morphology colony types. Urine samples collected from 50 cats with bacteriuria brought to Maeda Veterinary Hospital between
August 10, 2015 and March 31, 2017 (30 males and 20 females aged 0–19 years) were used in the study. Urine was collected bypuncture of the urinary bladder with a 25-G needle through the skin after confirming the position of the bladder in a supine position
with ultrasound. Urine was centrifuged and the precipitate was Gram-stained. Bacteriuria was diagnosed based on a positive Gram
stain.
Bacterial identification with the conventional method was performed using a MicroScan WalkAway system (Siemens Healthcare
Diagnostics, IL, U.S.A.). For the conventional culture, 1 µl of well-mixed urine was inoculated and spread on blood agar plates
using a sterile plastic disposable loop (Eiken Chemical Co., Tokyo, Japan). Plates were incubated in an aerobic atmosphere at
37°C for 18–24 hr. When bacterial growth was observed, the colonies on blood agar were counted, and colonies from both types ofplates were identified using the MicroScan WalkAway system.
DNA was extracted with a MagNA Pure Compact DNA isolation kit I (Roche Molecular Biochemicals, Mannheim, Germany).
Polymerase chain reaction amplification of the 16S rRNA gene was performed as described by Okazaki et al. [10] and Otsuka et al.
[13], using primers 8UA (5ˊ-AGA GTT TGA TC(A/C)TGG CTC AG-3ˊ) and 1485B (5ˊ-TAC GGT TAC CTT GTT ACG AC-3ˊ).
Amplicons were purified and sequenced using primers 519A (5ˊ-CAG C(A/C)G CCG CGG TAA T-3ˊ), 519B (5ˊ-ATT ACC GCG
GC(G/T)GCT G-3ˊ), 907A (5ˊ-AAA CT(T/C) AAA (T/G)GA ATT GAC GG-3ˊ), and 907B (5ˊ-CCG TCA ATT C(A/G)TTT (A/G)
A GTT T-3ˊ). A homology search of 16S rRNA gene sequences was performed against sequences registered in GenBank/EMBL/DDBJ using BLAST.
Urine sample preparation was performed using a MALDI Sepsityper kit (Bruker Daltonics, Bremen, Germany) [9]. In brief,
urine (5 ml) was centrifuged at 2,000 × g for 30 sec to remove leukocytes. The supernatant was centrifuged at 15,000 × g for
10 min to collect bacteria. The pellet in distilled water (1 ml) was transferred to a 1.5 ml reaction tube (Eppendorf, Hamburg,
Germany). After addition of 200 µl of lysis buffer, the sample was mixed using a vortex shaker for 10 sec and then centrifuged
for 1 min at 21,130 × g. The supernatant was discarded and the pellet was resuspended in 1 ml of washing buffer. After a second
centrifugation step (1 min at 21,130 × g), the supernatant was discarded and the pellet was suspended in 300 µl of distilled water.
After addition of 900 µl of ethanol, the sample was centrifuged at 21,130 × g for 2 min. Supernatant was discarded and residual
ethanol was removed after repeated centrifugation. The pellet was then suspended in 15 µl of 70% formic acid. Acetonitrile (15 µl)
was added and the sample was mixed using a vortex shaker and briefly centrifuged prior to MALDI-TOF MS analysis.
Urine sample preparation was also performed using the rapid BACpro kit (Nittobo Medical Co., Tokyo, Japan) [2]. In brief,
urine (5 ml) was centrifuged at 2,000 × g for 30 sec to remove leukocytes. The supernatant was then centrifuged at 15,000 × g
for 10 min to collect bacteria. The pellet samples were combined with 10 µl of reaction buffer 1, 100 µl of reaction buffer 2, and10 µl of polymer suspension in sequence. The next part of the protocol required use of a desktop centrifuge (Chibitan R, Merck
Millipore, Billerica, MA, U.S.A.). First, the mixture was separated by desktop centrifugation at 2,000 × g for 30 sec, and thesupernatant was discarded. Second, the resulting aggregate was resuspended in 1,000 µl of 70% acetonitrile, and the supernatant
was separated by centrifugation at 2,000 × g for 30 sec. Third, the obtained precipitate was resuspended in 30 µl of 70% formic
acid and 100 µl of 100% acetonitrile, and the supernatant was separated by centrifugation at 2,000 × g for 60 sec.
The MALDI-TOF α-cyano-4-hydroxycinnamic acid matrix was prepared daily as a saturated solution in 50% acetonitrileand 2.5% trifluoroacetic acid. Subsequently, 1 µl of the sample extract (prepared using either of the two approaches above) was
spotted on a steel target plate (Bruker Daltonik) and allowed to dry. Next, 1 µl of matrix solution was added and air dried. The
target plate was then placed in the MALDI-TOF MS apparatus. To identify the isolates, MALDI-TOF MS was performed on an
AutoFlex® TOF/TOF mass spectrometer equipped with FlexcontrolTM software v. 3.0 (Bruker Daltonics) for automatic acquisitionof mass spectra in the linear positive mode within a range of 2 to 20 kDa. Laboratory technicians performed all MALDI-TOF MS
measurements in the study.
The Autoflex® II TOF/TOF mass spectrometer was periodically calibrated using the Bruker Daltonics bacterial test standard(E.coli extracts containing RNase A and myoglobin). Calibration masses were as follows: RL36, 4364.3 Da; RS22, 5095.8 Da;
RL34, 5380.4 Da; RLmeth, 6254.4 Da; RL32, 6315.2 Da; RL29, 7273.5 Da; RS19, 10229.1 Da; RNase A, 13682.2 Da; andmyoglobin,16952.5 Da. Automated analysis of raw spectral data was performed using MALDI BioTyper automation v.3.0 software(Bruker Daltonics) with a library of 5,989 spectra (database updated on July 31, 2015) and default settings. The whole processfrom MALDI-TOF MS measurement to identification was performed automatically without user intervention. In brief, the software
generated a list of peaks up to 100. The threshold for peak acceptance was a signal-to-noise ratio of 10. After alignment, peaks witha mass-to-charge ratio difference of <250 ppm were considered to be identical. The peak lists generated were used for matches
against the reference library by using an integrated pattern matching algorithm in the software. Pattern-matching results are
expressed as scores ranging from 0 to 3, with a score <1.7 not considered to give reliable identification and a score ≥2.0 indicating
identification of a species [15].
To determine the minimal bacterial concentration allowing reliable MALDI-TOF MS identification, we inoculated 50-ml
aliquots of sterile water with one strain of E. coli or E. faecalis at a bacterial count of 2 × 108 CFU/ml. Sequential dilutions werethen used to achieve aliquots of each microorganism at counts of 1.0 × 107, 5.0 × 106, 1.0 × 106, 5.0 × 105, 1.0 × 105, 5.0 × 104 and1.0 × 104 CFU/ml. If necessary, aliquots were further diluted before spreading on a plate to obtain a countable number of colonies(50 to 500) when plated. Aliquots of 300 µl for each dilution were plated on blood agar plates, which were incubated at 37°C in an
aerobic atmosphere. Colonies were counted manually, and the mean of three aliquots at each dilution was considered to be the final
count for that dilution. Samples of 10 ml for each dilution were used for preparation for MALDI-TOF MS, as described above.
MALDI-TOF MS requires high bacterial counts to provide reliable scores. Inoculation with at least 1.0 × 105 CFU/ml was

doi: 10.1292/jvms.18-0145

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H. MAEDA ET AL.

Table 1. MALDI-TOF MS scores for different Escherichia coli and Enterococcus faecalis bacteria counts
Kit

Microorganism

MALDI-TOF MS score for bacteria count (CFU/ml)
1.0 × 107

5.0 × 106

1.0 × 106

5.0 × 105

1.0 × 105

5.0 × 104

1.0 × 104

MALDI Sepsityper E. coli
E. faecalis

2.321
2.122

2.301
2.115

2.284
1.986

2.184
1.862

2.105
1.792

1.988
1.662

1.904
1.624

E. coliE. faecalis

2.398
2.182

2.321
2.182

2.299
2.162

2.186
2.084

2.134
2.051

1.991
1.742

1.972
1.722

Rapid BACpro

Table 2. Urine culture results of 39 specimens analyzed by MALDI-TOF MS
Urine culture(No. of isolates)
Growth of colonies (N=37)
1-colony morphology (N=29)2-colony morphology (N=8)

MALDI sepsityper(No. of isolates)

Rapid BACpro(No. of isolates)

Positive with same identification (25)a)
Not reliable identification (4)b)
Not reliable identification (8)Positive with same identification (25)a)
Not reliable identification (4)b)
Enterococcus faecalis and Citrobacter freundii (2)Enterococcus faecalis and Enterobacter cloacae (2)Enterococcus faecalis and Klebsiella pneumoniae (3)Not reliable identification (1)c)

No growth (N=13)a) A total of 25 correct identifications at the species level. b) Four cases were cultures with <1.0 × 105 CFU/ml. c) Cases of E. faecalis andE. cloacae were cultures with <1.0 × 105 CFU/ml.

Table 3. Identification by MALDI-TOF MS of 16S rRNA and conventional identification of 45 microorganisms causing cystitis
with bacterial counts ≥1.0 × 105 CFU/ml
Conventional method(No. of isolates)
Citrobacter freundii (2)Enterobacter cloacae (4)Escherichia coli (22)Klebsiella pneumoniae (3)Total gram-negative bacteria (31)

MALDI-TOF MS method
MALDI sepsityper kit(No. of isolates)
No reliable identification (2)Enterobacter cloacae (3)No reliable identification (1)Escherichia coli (22)Klebsiella pneumoniae (3)90.3% (28/31)

Enterococcus faecalis (9)No reliable identification (9)Staphylococcus aureus (5)Staphylococcus aureus (3)No reliable identification (2)Rapid BACpro(No. of isolates)
Citrobacter freundii (2)Enterobacter cloacae (3)No reliable identification (1)Escherichia coli (22)Klebsiella pneumoniae (3)96.7% (30/31)
Enterococcus faecalis (8)No reliable identification (1)Staphylococcus aureus (3)No reliable identification (2)16S rRNA method(No. of isolates)
Citrobacter freundii (2)Enterobacter cloacae (4)Escherichia coli (22)Klebsiella pneumoniae (3)100.0% (31/31)
Enterococcus faecalis (9)Staphylococcus aureus (5)Total gram-positive cocci (14)21.4% (3/14)

78.5% (11/14)

100.0% (14/14)

Total (45)68.8% (31/45)

91.1% (41/45)

100.0% (45/45)

required to obtain reliable scores for E. coli after preparation with the MALDI Sepsityper Kit and rapid BACpro, and the required
level was higher for E. faecalis using the MALDI Sepsityper Kit (5.0 × 106 CFU/ml) and rapid BACpro (1.0 × 105 CFU/ml)(Table 1). Of the 50 urine specimens, growth of colonies was observed in 37 specimens: 29 with single-colony morphology, and8 with two-colony morphology. Thirteen specimens did not grow in culture, and MALDI-TOF MS did not identify a significant
protein profile in any of these cases (Table 2).
In specimens with single-colony morphology, MALDI-TOF MS correctly identified 25 isolates (86.2%) using the MALDI
Sepsityper Kit and 25 isolates (86.2%) with rapid BACpro. In the 4 cases in which identification was not possible, the bacterial
count was ≤1.0 × 105 CFU/ml, which was below the detection limit. In specimens with two-colony morphology, MALDI-TOF MS
gave correct results in only one specimen (Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae) using the MALDI
Sepsityper Kit, whereas with rapid BACpro 2 species were detected in 7 urine specimens. No bacterium were identified with rapid
BACpro in one case, in which E. faecalis and E. cloacae were present and the E. cloacae count was 1.0 × 105 CFU/ml, which was
below the detection limit.
Thirty-two strains were identified using the conventional method, including 31 enteric Gram-negative bacteria (GNB) and
14 gram-positive cocci (GPC) (Table 3). MALDI-TOF MS correctly identified 28 enteric GNB (90.3%) and 3 GPC (21.4%)

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with the MALDI Sepsityper Kit, and 29 enteric GNB (96.7%) and 11 GPC (78.5%) with rapid BACpro. Overall, among the
45 specimens with colony counts ≥1.0 × 105 CFU/ml, microorganism identification coincided with the MALDI Sepsityper
Kit in 31 cases (68.8%) and rapid BACpro in 40 cases (91.1%) (Table 3). Of the GNB, C. freundii, E. cloacae, E. coli, and K.
pneumoniae were correctly identified in 0 (0.0%) and 2 (100.0%), 3 (75.0%) and 3 (75.0%), 22 (100.0%) and 22 (100.0%), and3 (100.0%) and 3 (100.0%) samples using the MALDI Sepsityper Kit and rapid BACpro, respectively. Of the GPC, E. faecalis
and S. aureus were correctly identified in 0 (0.0%) and 8 (88.8%), and 3 (60.0%) and 3 (60.0%) using the MALDI Sepsityper Kit
and rapid BACpro, respectively. The E. faecalis strain, which could not be identified, was present at 1.0 × 105 CFU/ml, which
was below the detection limit. The 45 strains identified by the conventional method were subjected to 16S rRNA analysis, andidentification was consistent for all strains.
Urine from animals with LUTD contains many white and red blood cells and bacteria. In pretreatment, urine samples were
centrifuged at 2,000 × g for 30 sec to remove white blood cells, and then at 14,100–15,500 × g for 5–10 min to collect bacteria[5, 8]. A sufficient bacterial count is essential for identification by MALDI-TOF MS, and our findings support a previous reportshowing that the bacterial count needed for good discrimination by MALDI-TOF MS is higher for GPC (2.5–5 × 105 CFU/ml) thanfor GNB (about 6 × 104 CFU/ml) [17]. Rapid BACpro improved the collection, quantity and purity of the collected bacteria. The
E. faecalis identification rate was 0% using the MALDI Sepsityper Kit, but 88.8% using rapid BACpro, and the GPC identification
rate was 24.1% using the MALDI Sepsityper Kit, but 78.5% using rapid BACpro. The bacteria harvesting efficiency of rapid
BACpro was high and this improved the identification rate. The concordance rate of the two-morphology colony-type urine
samples was 31.8–60.0%, and 2 species could be identified.
We attempted to improve the identification rate by using pretreatment kits for direct bacterial identification from positive blood
culture bottles: MALDI Sepsityper Kit and rapid BACpro. The MALDI Sepsityper Kit collects bacteria by lysing blood cells ina lysis buffer [14], whereas rapid BACpro isolates bacteria from urine through selective binding of bacteria to a cationic polymer
suspension [2]. The concordance rates of identification using MALDI-TOF MS with pretreatment with the MALDI Sepsityper
Kit and rapid BACpro relative to the conventional method were 68.8 and 91.1%, respectively (Table 3). These rates are similar to
those reported previously [5]. The concordance rates for identification of E. faecalis and E. cloacae by MALDI-TOF MS using
rapid BACpro were 88.8% and 75.0%, respectively, showing improvement of these rates. The E. faecalis detection sensitivity of
MALDI-TOF MS using rapid BACpro was 1.0 × 105 CFU/ml (Table 1), and the E. faecalis count was 1.0 × 105 CFU/ml, which
was below the detection limit. The limit of detection of E. cloacae was also 1.0 × 105 CFU/ml, which is a low bacteria count. Thus,
MALDI-TOF MS identification of bacteria using rapid BACpro pretreatment is possible at a bacterial level ≥1.0 × 105 CFU/ml.
In two-morphology colony-type urine samples, the concordance rates for MALDI-TOF MS using the MALDI Sepsityper Kit andrapid BACpro relative to the conventional method were 0.0 and 87.5%, respectively (Table 2). The one non-identifiable case rapid
BACpro contained E. faecalis and E. cloacae, and the E. cloacae count was low (1.0 × 105 CFU/ml). MALDI-TOF MS spectraof the two-morphology colony types were complex and the samples contained components other than bacteria, which reduced
the concordance rate. Since the MALDI Sepsityper Kit lyses blood cells with lysis buffer and collects bacteria, complete removalof blood cell components is difficult. In contrast, samples treated with rapid BACpro contain very few non-bacterial components
because this method isolates bacteria through selective binding of bacteria to a cationic polymer suspension without lysing blood
cells [2].
Urine culture (i.e., quantitative culture of urine specimens on solid medium followed by biochemical characterization ofisolates), which is the gold standard for diagnosis, requires 24–72 hr until the results are available [3]. In contrast, MALDI-TOF
MS of urine specimens prepared using rapid BACpro and the MALDI Sepsityper Kit can identify bacterial species using MALDI
BioTyper automation software within about 30 min, and rDNA analysis requires about 1 hr. The transition from conventional
microbiological methods to MALDI-TOF MS also resulted in significant laboratory cost savings, estimated to be 51.7% of total
costs during a 12-month study [16].
In conclusion, MALDI-TOF MS using rapid BACpro for pretreatment is a quick and reliable method for identification ofbacteria in infected urine. The shortened analysis time enables earlier and more accurate selection of antibiotics for treatment of
feline bacteriuria.
ACKNOWLEDGMENTS. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI C: 16K08979) and a
research project grant awarded by Azabu University.
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