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MALDI-TOF MS IDENTIFICATION of CITROBACTER YOUNGAE ISOLATED from FOOD Dubravka S

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MALDI-TOF MS IDENTIFICATION of CITROBACTER YOUNGAE ISOLATED from FOOD Dubravka S DOI: 10.5937/FFR1802107M UDK 579.84:616.98]:637.3 Short communication MALDI-TOF MS IDENTIFICATION OF CITROBACTER YOUNGAE ISOLATED FROM FOOD Dubravka S. Milanov*1, Milan D. Đilas2, Jelena М. Babić1, Bojana Z. Prunić1, Maja Ј. Velhner1 1 Scientific Veterinary Institute ‘Novi Sad’, Rumenački put 20, 21000 Novi Sad, Serbia 2 Institute of Public Health of Vojvodina, Futoška 121, Novi Sad, Serbia *Corresponding author Tel.: +381 21 4895 346 E-mail: [email protected] ABSTRACT: Salmonella-like bacteria, such as certain species of the Citrobacter genus are sometimes isolated from food and feed. By testing their biochemical and, partially, serological characteristics (according to the regulations given in the ISO 6579-1:2017 Standard) it is impossible to completely reject their affiliation to the Salmonella genus. In addition to this, the food microbiology laboratories usually lack diagnostic tools and procedures for precise identification of these isolates to the species level. Thus, it is considered useful to describe the identification of Citrobacter youngae species using MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). Besides differential diagnostic criteria for distinguishing between Salmonella spp. and Citrobacter youngae, in this communication the relevant data on Citrobacter spp., the emerging opportunistic pathogenic member of Enterobacteriaceae family were presented. Key words: Salmonella-like bacteria, cheese A REPORT ON A LABORATORY CASE A sample of soft, medium-fat cheese accordance with the ISO 6579-1:2017 sprinkled with spices, produced in a small Standard (Table 1), plus using the methyl- scale plant, was examined for the pre- red and citrate utilization tests, which were sence of Salmonella spp. in the Laboratory both positive (Figure 2). of Food Microbiology of the Scientific Veterinary Institute in Novi Sad. The sam- Rapid slide agglutination test with the poly- ple was tested in accordance with the valent serum for Salmonella groups A, B, requirements of the ISO 6579-1:2017 C, D and E (Institute for Public Health of Standard. Characteristic pink colonies with Serbia, Belgrade, Serbia) was positive but a black centre of Salmonella species on not with the group-specific sera B (O:4), XLD agar (Biokar Diagnostics, France) D1 (O:9), C1 (O:7), C2-C3 (O:8), B (O:4) were isolated from the sample and futher and E1 (O:3,10). examined. Based on the negative reaction of lysine With tests of catalase (positive) and oxi- decarboxylase and the production of -ga- dase (negative) it was confirmed that the lactosidase, with 95% and 99% probabi- isolate belonged to the Enterobacte- lity, respectively, the hypothesis that the riaceae family. The biochemical charac- isolate belonged to Salmonella genus was teristics of the isolate were assessed in ruled out. Dubravka S. Milanov et al., MALDI-TOF MS identification of Citrobacter youngae isolated from food, Food and Feed Research, 45 (2), 107-112, 2018 A. Bacterial colonies isolated from the cheese B. Colonies of the reference Salmonella sample Enteritidis ATCC 13076 culture Figure 1. Colonies on XLD agar medium after 24h incubation at 37 C C. Isolate from the cheese sample D. Positive control: Salmonella Enteritidis ATCC 13076 From left to right: Kligler, citrate, urea, lysine decarboxylase, methyl red, indole, -galactosidase (ONPG disk) Figure 2. Results of biochemical testing Table 1. Results of biochemical tests Isolate Other strains* Test Reaction %** TSI*** acid from glucose + + 100 TSI gas from glucose - + 92 TSI acid from lactose - - 1 TSI hydrogen sulfide produced + + 92 Urea hydrolysis - - 1 Lysine decarboxylation - + 95 Production of indole - - 2 -galactosidase + - 1 *Interpretation of biochemical tests for other Salmonella strains (does not refer to the following species: S. typhi, S. paratyphi A, B and C; S. Gallinarum biovar gallinarum and biovar pullorum); **Percentages indicate that not all isolates of Salmonella serovars show the reactions marked + or -. Reaction may also vary between and within serovars; ***TSI-Triple sugar iron (Source: ISO 6579-1:2017,p.10) Dubravka S. Milanov et al., MALDI-TOF MS identification of Citrobacter youngae isolated from food, Food and Feed Research, 45 (2), 107-112, 2018 6257.156 Intens. [a.u.] Intens. 5215.929 8000 4365.215 6000 3126.356 4000 2702.861 4778.685 7092.525 3544.635 9557.193 2000 2178.563 3934.686 7871.648 9198.405 8371.431 6600.292 10287.941 10069.308 11354.361 0 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 m/z Figure 3. Spectra of Citrobacter youngae isolate generated by the MALDI-TOF Bruker Biotyper. The x- axis shows the masses (m/z) of the ions, and the y-axis shows the absolute intensities of the ions. The m/z values represent the mass-to-charge ratio Based on the positive test for glucose the control of flexControl software ver. 3.4 fermentation, citrate utilization test, methyl (Bruker Daltonics). Spectra in the mass red and -galactosidase activity, as well as range of 2 to 20 kDa were collected by ac- the characteristic appea-rance of the co- cumulating 240 laser shots (laser fre- lonies on the XLD agar medium, the quency, 60 Hz; ion source 1 voltage, 20 diagnostic assumption was that the isolate kV; ion source 2 voltage, 18.15 kV; lens belonged to Citrobacter genus. Moreover, voltage, 6 kV) at 30–40% of maximum the bacterial culture produced a putre- laser power. faction smell, which is not characteristic of Salmonella species. In order to identify the COMMENT species, the isolate was further processed The most important food borne pathogens at the Institute for Public Health of Voj- belong to the Enterobacteriaceae family. vodina in Novi Sad, Serbia. These are Salmonella spp., Yersinia ente- MALDI-TOF MS analysis rocolitica, pathogenic Escherichia coli and Shigella spp. Some members of the fa- The isolate was prepared using the stan- mily, for instance, Providencia alcalifa- dard Bruker’s direct transfer sample pre- ciens, Klebsiella spp., Serratia spp. and paration procedure for MALDI-TOF MS. A Citrobacter spp., are emerging opportu- single bacterial colony was spotted directly nistic pathogens (Baylis et al., 2011). Ba- onto a MALDI target plate (Bruker Dalto- sed on the results of DNA hybridization nics, Germany), allowed to dry and imme- assay, Citrobacter are classified into 11 diately overlaid with 1.0 μL of the matrix species: C. amalonaticus, C. braakii, C. solution (Bruker Matrix HCCA; α-Cyano-4- farmeri, C. freundii, C. gillenii, C. koseri, C. hydroxycinnamic acid). murliniae, C. rodentium, C. sedlakii, C. MALDI-TOF mass spectra were obtained werkmanii and C. youngae (Brener et al., using Microflex LT/SH Biotyper spectro- 1993). Citrobacter spp. are typically motile meter (Bruker Daltonics, Germany) equip- bacteria, owing the peritrichous flagellae, ped with a nitrogen laser (337 nm) under facultative anaerobic, usually oxidase ne- Dubravka S. Milanov et al., MALDI-TOF MS identification of Citrobacter youngae isolated from food, Food and Feed Research, 45 (2), 107-112, 2018 gative, and positive for catalase and MALDI-TOF MS in the iden-tification of methyl red (Doran, 1998; Duman et al., Citrobacter species in food samples origin- 2017). They generally utilize citrate as the nating from pork (Kwak et al., 2015). only carbon source, which explains the Within less than a decade MALDI-TOF MS etymology of the name of the genus has become a gold standard in clinical (Arens et al., 1996). Two additional pro- microbiology laboratories, as it represents perties of citrobacteria which help to a powerful, rapid, precise, and cost-ef- distinguish them from other members of fective method for identification of bac- the family Enterobacteriaceae are their teria, mycobacteria, and fungi, compared failure to produce acetylmethylcarbinol to conventional phenotypic techniques or (negative Voges-Proskauer test) and the molecular biology. lack of lysine decarboxylase (Janda et al., 1994). MALDI-TOF MS is able to detect a large spectrum of proteins, allowing the discri- Citrobacter species are present in water, mination between closely related species soil, food and the intestinal tracts of and identification at the species and sub- animals and humans (Doran, 1998; Baylis species levels (Biswas and Rolan, 2012; et al., 2011; Clermont et al., 2015), and Murray, 2012). are commonly recovered from human faecal/intestinal flora (Duman et al., 2017). This technique allows desorption of pep- They can cause urinary infections, hos- tides and proteins from both whole diffe- pital-acquired bacteremiae, brain abscess- rent cultured bacteria and crude bacterial ses, neonatal meningitis, wound infec- extracts (Biswas and Rolan, 2012). tions, pneumoniae, abscess, septicae- Usually, a single bacterial colony is miae, endocarditis and diarrhoea (Arens et enough for MALDI-TOF MS analysis (Dek- al., 1996; Doran, 1998; Clermont et al. ker and Branda, 2011). Bacterial colonies 2015). Citrobacter spp. are also conside- are removed from agar culture plates, red to be capable of causing gastrointes- mixed with a matrix compound, and dried tinal infections especially in infants and on steel target plates. The dried prepa- young children (Janda et al., 1994). rations are exposed to laser pulses, re- An outbreak of haemolytic uraemic syn- sulting in energy transfer from the matrix drome (HUS) in Germany was linked to to the nonvolatile analyte molecules, with the consumption of butter containing par- desorption of analyte into the gas phase. sley contaminated with C. freundii carrying The ionized molecules are accelerated by the vtx2 gene (Tschape et al., 2011). The electric potentials through a flight tube to infections most often occur in neonates the mass spectrometer, with separation of and immunocompromised people (Thurm the biomarkers determined by their and Gericke, 1994; Doran, 1998). C. mass/charge ratio (m/z). The profile of freundii and C. koseri are the two most biomarkers is then compared with profiles common clinical isolates. The other nine of a collection of well characterized orga- Citrobacter species, including C.
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