Characterization of Klebsiella oxytoca using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system

Abstract

Background: Antibiotic-associated Hemorrhagic colitis (AAHC) is an inflammation of the intestine occurs followed by antibiotic treatment and has been related to Klebsiella oxytoca in recent studies. In this generation, the identification of bacteria could be rapid and reliable with the Matrix Assisted Laser Desorption Ionization Time- Of-Flight Mass Spectrometry (MALDI-TOF MS) technology. However, there were very few studies in investigating K. oxytoca and its cytotoxin producing ability utilizing MALDI-TOF.

Aim: The aim of this study is to reveal more information of K. oxytoca infection within the hospitalized patients, explore the identification performance and search for unique cytotoxin positive protein pattern of K. oxytoca utilizing MALDI-TOF MS.

Method: K. oxytoca isolates were collected between 2009 to 2014 from Queen Mary Hospital (HK). The isolates were collected from stool, peripheral blood, urine, bile and miscellaneous samples. All isolates were challenged by conventional biochemical tests and MALDI-TOF MS. Toxigenicity of the isolates were assessed by cytotoxin neutralization assay. Spectra of the selected isolates were analysed within m/z range 2,000 Da-15,000 Da. The protein profiling and dendrogram was achieved utilizing MALDI Biotyper 3.1 software.

Results: Among the 91 K. oxytoca isolates, 41 (45%) isolates was cytotoxin positive. Stool isolates showed the highest proportion 15/29 (52%) followed by peripheral blood 15/34 (44%), urine and miscellaneous at 5/12 (42%) and bile culture at 1/4 (25%). (p = 0.86).

The ratio of male/ female carriers was 1:1.07. The proportion of cytotoxin positive strains of male carrier was 17/44 (38.6%) and 24/47 (51.1%) on female carrier. (p = 0.23).

The proportion of cytotoxin positive strains among the 0-18 age group was 4/12 (33.3%), age 19-65 was 20/44 (45.5%) and age above 65 was 17/35 (48.6%). (p = 0.66).

The proportion of cytotoxin positive on patient with digestive system related diseases was 14/29 (48.3%), liver related diseases was 4/6 (66.6%), fever was 4/6 (66.6%), chest infection was 2/6 (33.4%) and others was 17/44 (38.6%). (p=0.49).

The proportion of cytotoxin positive of normal stool was 11/24 (45.8%) and abnormal stool was 4/19 (21.0%). (p=0.09). MALDI-TOF MS Identification: It was not able to identify K. oxytoca at species level. The turn-around-time for identifying K. oxytoca with MALDI-TOF MS was reduced by 45%.

Mass spectrum analysis: K. oxytoca isolates originated from stool specimen with the unique protein pattern 10/15 (66.6%) (3 distinctive protein peaks at 3847 Da, 6822 Da and 7686 Da) had shown a significant association with cytotoxin-producing ability (p=0.00016).

Cluster analysis: Isolates was branched into 5 clusters according to the distant level of the protein spectrum. The association of the cytotoxin-production ability and the cluster was significant (p=0.001).