Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C 4 plant, Pennisetum glaucum

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Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C 4 plant, Pennisetum glaucum

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  ORIGINAL PAPER Biochemical and molecular characterization of   Cronobacter  spp  .  (formerly  Enterobacter sakazakii ) isolated from foods Imrich Turcovsky´  • Kristı´na Kunikova´  • Hana Drahovska´  • Eva Kaclı´kova´ Received: 1 March 2010/Accepted: 6 July 2010/Published online: 17 July 2010   Springer Science+Business Media B.V. 2010 Abstract  The aim of this study was to identify andcharacterize  Cronobacter   spp. isolated from a rangeof foods. A total of 71  Cronobacter   strains wereisolated from 602 foods in our laboratory. Thehighest contamination was observed in foods of plantsrcin, e.g. spices, teas, chocolate, nuts, pastries andvegetables. On the basis of genus and speciesidentification performed using genus-specific PCR,16S rRNA sequencing and AFLP genotyping, mostof the strains belonged to  Cronobacter sakazakii .Biochemical profiling by the tests included inAPI 20E, complemented with relevant additionaltests, classified the strains into 13 biogroups. AFLPgenotyping facilitated discrimination of six maingroups at the 70% similarity level and strain groupingcorrelated clearly with species identification. Ourresults indicate that molecular typing by AFLP maybe applied as a useful tool not only for directcomparison of   Cronobacter   isolates, providing trace-ability, but also for the reliable species classification.Moreover, tracing of these bacteria in a wider varietyof foods should be important to enhance the knowl-edge of their transmission. Keywords  Cronobacter     Characterization   Food isolates    Biotyping    AFLP genotyping Introduction Enterobacter sakazakii  previously referred to as‘yellow pigmented  E. cloacae ’, was defined as a newspecies in 1980 (Farmer et al. 1980) and 15 biogroupswere described based on biochemical character-ization. Members of this species were consideredrelatively phenotypically and genotypically hetero-geneous (Lehner et al. 2004), a 16th biogroup hasbeen reported and the existence of several geneticgroups has been demonstrated based on 16S rRNAgene sequence analysis (Iversen et al. 2006).  E. sak-azakii  strains were divided into separate groupsbased on f-AFLP fingerprints, ribopatterns and full-length 16S rRNA sequences. DNA–DNA hybridiza-tion revealed several genomospecies (Iversen et al.2007) and, subsequently, led to classification of thesebacteria into six species within the new  Cronobacter  genus (Iversen et al. 2008a). Subsequently, the six Cronobacter   species were supported by multilocussequence analysis (Kuhnert et al. 2009; Baldwin et al.2009) and a differentiation system based on PCRtargeting the  rpoB  gene was developed (Stoop et al. I. Turcovsky´    E. Kaclı´kova´ ( & )Department of Microbiology and Molecular Biology,Food Research Institute, PO Box 25,82475 Bratislava, Slovakiae-mail: kaclikova@vup.sk K. Kunikova´    H. Drahovska´Department of Molecular Biology, Faculty of NaturalSciences, Comenius University, Bratislava, Slovakia  1 3 Antonie van Leeuwenhoek (2011) 99:257–269DOI 10.1007/s10482-010-9484-7  2009). The whole genome sequence of the  Cronob-acter sakazakii  ATCC BAA-894 has been recentlypublished and its genome was compared with repre-sentatives of all  Cronobacter   species by microarrayanalysis. It was shown that about 55% of genes werecommon to  C. sakazakii  strains and 43% werecommon to all  Cronobacter   strains (Kucerova et al.2010). Cronobacter   spp .  are considered to be opportunis-tic pathogens, implicated in particularly severe food-borne diseases in neonates and infants (Farber 2004;Mullane et al. 2007; Townsend et al. 2008). Symp- toms include bacteraemia, necrotizing enterocolitisand meningitis, with fatality rates as high as 80%(Lai 2001). Although these microorganisms are widelydistributed in the environment (Kandhai et al. 2004),dried infant milk formula has been implicated as thevehicle of transmission in many clinical manifesta-tions (Nazarowec-White and Farber 1997; van Ackeret al. 2001; Gurtler et al. 2005). More recently, infections in adults have been reported, in particularamong the elderly and immunocompromised patients(Ray et al. 2007; See et al. 2007). More than 500 Cronobacter   infections were reported in UnitedKingdom during 1999–2007; 90% of them occurredin persons older than 15 years (FAO/WHO report2008). For persons with a decreased immunity, inparticular neonates, elderly and persons with severeunderlying diseases, the occurrence of   Cronobacter  spp. in food and environment may represent a hazardto health (Lai 2001; Friedemann 2007). The primary reservoirs for subsequent food con-tamination remain undefined due to the ubiquitousnature of these bacteria.  Cronobacter   strains wereisolated from a wide range of foods including milk,cheese, dried foods, meats, water, vegetables, rice,tea, herbs and spices (Iversen and Forsythe 2004;Edelson-Mammel et al. 2005; Friedemann 2007), various food production environments and house-holds (Kandhai et al. 2004; Gurtler et al. 2005). The spectrum of   Cronobacter  -contaminated foods coversboth raw and processed foods and the kind of processing is not restricted to dry products (Friede-mann 2007).Isolation and detection of   Cronobacter   spp. isbased on standardised microbiological procedures of enrichment followed by the isolation of colonies onchromogenic media. Confirmation of these coloniesis necessary to verify the identity of the isolates.Commercial biochemical systems or PCR-basedassays are available for food and clinical microbio-logical laboratories (Fanjat et al. 2007; Stoop et al.2009; Druggan and Iversen 2009). Generally, pheno- type analysis may provide unreliable identification aslong as it is based on unstable expression of themarkers (Drudy et al. 2006; Druggan and Iversen2009).DNA-based methods offer an alternative approach,enabling a direct comparison of isolates in outbreaks.Molecular methods may facilitate the trace back of outbreak isolates from clinical samples to the sourceof contamination and provide useful tools to targetcontrol strategies to reduce the risk of transmission(Drudy et al. 2006).In this study, 98 strains belonging to the genus Cronobacter  , including 71 strains isolated in ourlaboratory, were identified and characterized usingbiochemical profiling and molecular techniques,namely genus-specific real-time PCR, AFLP typingand partial 16S rRNA sequencing. Since  Cronobact-er   spp. are ubiquitous in many environments, a widerange of foods was analysed. Materials and methods Isolation of   Cronobacter   spp. from food Cronobacter   strains were isolated from various foodsamples analysed within the frame of a surveillanceprogramme for coliforms in the State Veterinary andFood Institute, Bratislava, Slovakia in 2006–2009(Table 1). Strains were isolated from 5 replicates of a25 g test portion of each food sample according to thestandard method specified in ISO/TS 22964:2006using the two-step enrichment consisting of pre-enrichment (1/10) in Buffered Peptone Water (Merck,Darmstadt, Germany) and selective enrichment(1/100) at 45  C in modified Lauryl Sulfate Tryptose(mLST) broth (Merck) with additional NaCl (Merck)to the final concentration of 0.5 mol l - 1 and vanco-mycin 10 mg l - 1 (Fluka, Buchs, Switzerland). Pre-sumptive  Cronobacter   strains were isolated as typicalgreen–blue colonies grown on Chromogenic Entero-bacter sakazakii agar, DFI formulation (Oxoid,Basingstoke, England) or Chromocult Enterobac-ter sakazakii (Merck) based on the expression of  a -glucosidase activity. Identification of   Cronobacter  258 Antonie van Leeuwenhoek (2011) 99:257–269  1 3  spp. was confirmed by real-time PCR targeting the dnaG  gene according to Seo and Brackett (2005).Our collection of strains was supplementedwith 22 strains from the American Type CultureCollection (ATCC, Manassas, VA, USA), BelgianCo-ordinated Collections of Microorganisms in Gent(BCCM/LMG Bacteria Collection) and from CzechCollection of Microorganisms (CCM, Brno, CzechRepublic). Five further strains isolated from fruitpowder, kindly provided by Professor Roger Stephanfrom the Institute of Food Safety and Hygiene (ILS),Vetsuisse Faculty University of Zurich, Switzerland,were also added to the collection. All strains weremaintained in 20% glycerol solution at  - 18  C orfreeze-dried for long-term storage. Bacteria werecultured in Luria–Bertani or Brain Heart broth, andon Brain Heart agar (Merck) at 37  C. Strains of  Cronobacter   spp. used in the study are listed inTable 2.Phenotypic characterization and biotypingProduction of the yellow pigment was tested onTryptone Soya Agar (Oxoid) supplemented with bilesalts (Fluka) with exposure to light during incubation.In addition to the results derived from biochemicalreactions included in the biochemical test kit API 20E(bioMe´rieux, Marcy l’Etoile, France), the followingtests were performed using conventional methods.Tests for the acid production from carbohydrates wereexamined in broth base (peptone 10 g l - 1 , yeastextract 1 g l - 1 and NaCl 5 g l - 1 ) with phenol redindicator (0.018 g l - 1 ). Filter-sterilized dulcitol andmethyl- a - D -glucopyranoside (both from Fluka), at a Table 1  Occurrence of   Cronobacter   spp. in analysed food categoriesOrigin Sample category Number of analysedsamplesNumber of positive samplesSpeciesPlant Spices 21 13  C. sakazakii ,  C. malonaticus , C. muytjensii Tea 5 3  C. sakazakii Chocolate products 37 11  C. sakazakii ,  C. malonaticus Pastries 9 5  C. sakazakii ,  C. malonaticus Nuts 2 2  C. sakazakii Cereals and derived products 20 0Instant soups 13 2  C. malonaticus ,  C. dublinensis Fruits and derived products 12 0Vegetables 12 5  C. sakazakii ,  C. turicensis , C. muytjensii Altogether 131 41Animal Raw meat 64 1  C. sakazakii Raw meat (spiced or marinated) 48 17  C. sakazakii ,  C. malonaticus , C. dublinensis , ni.Meat semiproducts 29 0Meat products (ready-to-eat) 81 9  C. sakazakii ,  C. malonaticus Eggs and derived products 51 0Milk powder and derived products 55 1  C. malonaticus Milk and milk products 98 0Cheese 32 0Raw meat swabs 13 1  C. sakazakii Altogether 471 29Environmental Dust 2 1  C. sakazakii Altogether 604 71Antonie van Leeuwenhoek (2011) 99:257–269 259  1 3  Table 2  Biochemical and molecular identification of   Cronobacter   strains used in this studyNo Designation Source API 20E % id. Cronobacter Biogroup AFLP Species1 ATCC 29544 a Human 3305373 98.40 1 A  C. sakazakii 2 ATCC 12868 a Human 3305373 98.40 3 A  C. sakazakii 3 ATCC 29004 Unknown 3305373 98.40 3 A  C. sakazakii 4 ATCC BAA 894 a Infant formula 3305173 51.10 2 A  C. sakazakii 5 LMG 2758 Human 3305173 51.10 2 A  C. sakazakii 6 LMG 2759 a Human 3305173 51.10 2 A  C. sakazakii 7 LMG 2760 Brush 3305173 51.10 2 A  C. sakazakii 8 LMG 2762 Human 3305173 51.10 2 A  C. sakazakii 9 LMG 2763 Human 3305173 51.10 2 A  C. sakazakii 10 LMG 2766 Human 3305373 98.40 1 A  C. sakazakii 11 LMG 2786 a Human 3305373 98.40 1 A  C. sakazakii 12 CCM 3460 Human 3305373 98.40 1 A  C. sakazakii 13 CCM 3479 Human 3304373 98.40 13 A  C. sakazakii 14 ILS 744/03 a Fruit powder 3305373 98.40 1 A  C. sakazakii 15 ILS 759/03 Fruit powder 3305373 98.40 1 A  C. sakazakii 16 ILS 954/03 Fruit powder 3304373 97.30 13 A  C. sakazakii 17 ILS 1094/04 Fruit powder 3305173 51.10 2 A  C. sakazakii 18 ILS 1155/04 a Fruit powder 3305373 98.40 1 A  C. sakazakii 19 121206/23 a Ginger 3305373 98.40 1 A  C. sakazakii 20 121206/24 Black pepper 3305373 98.40 1 A  C. sakazakii 21 121206/27 Black pepper 3305373 98.40 1 A  C. sakazakii 22 201206/17 Chocolate 3305173 51.10 7 A  C. sakazakii 23 201206/18 a Chocolate 3305173 51.10 3 A  C. sakazakii 24 201206/19 a Chocolate 3205373 98 4 A  C. sakazakii 25 201206/22 a Chocolate 3205373 98 4 A  C. sakazakii 26 201206/23 Chocolate 3305373 98.40 3 A  C. sakazakii 27 201206/25 a Chocolate 3305173 51.10 2 A  C. sakazakii 28 240107/09 Hazelnut 3305373 98.40 1 A  C. sakazakii 29 210307/02 Pastries 3305173 51.10 2 A  C. sakazakii 30 210307/06 Pastries 3305173 51.10 2 A  C. sakazakii 31 210307/11 Pastries 3305373 98.40 1 A  C. sakazakii 32 210307/19 a Chicken liver 3205373 98 4 A  C. sakazakii 33 040407/76 Sausage 3305373 98.40 1 A  C. sakazakii 34 170407/02 Hot chocolate 3305373 98.40 8 A  C. sakazakii 35 170407/05 Fruit tea 3205373 98 4 A  C. sakazakii 36 170407/07 Herbal tea 1305373 82.10 1 A  C. sakazakii 37 170407/10 Black tea 3305373 98.40 1 A  C. sakazakii 38 240407/26 a Marinated meat 3305373 98.40 1 A  C. sakazakii 39 150507/78 Meat product 3305373 98.40 1 A  C. sakazakii 40 120607/02 Nut chocolate 3307173 98.80 4 A  C. sakazakii 41 120607/66 Nut chocolate 3307173 98.80 1 A  C. sakazakii 42 270607/29 Chicken pate´ 3305173 51.10 2 A  C. sakazakii 43 270607/30 Chicken pate´ 3305373 98.40 1 A  C. sakazakii 44 270607/31 Meat pate´ 3305373 98.40 1 A  C. sakazakii 45 100707/19 Peanuts 3305373 98.40 1 A  C. sakazakii 260 Antonie van Leeuwenhoek (2011) 99:257–269  1 3  Table 2  continuedNo Designation Source API 20E % id. Cronobacter Biogroup AFLP Species46 140807/15 Marinated meat 3305373 98.40 1 A  C. sakazakii 47 210807/15 Meat pate´ 3305373 98.40 1 A  C. sakazakii 48 210807/25 Black pepper 3305373 98.40 1 A  C. sakazakii 49 210807/31 Red pepper 3305373 98.40 1 A  C. sakazakii 50 210807/39 Marinated meat 3305173 51.10 2 A  C. sakazakii 51 110907/09 Black pepper 3305373 98.40 1 A  C. sakazakii 52 110907/13 Black pepper 3305373 98.40 1 A  C. sakazakii 53 250907/17 Marinated meat 3305373 98.40 1 A  C. sakazakii 54 091007/14 a Marinated meat 3305373 98.40 1 A  C. sakazakii 55 161007/37 Meat product 3305373 98.40 1 A  C. sakazakii 56 061107/07 a Red pepper 1205363 0.00 4 A  C. sakazakii 57 061107/08 a Marinated meat 3305373 98.40 1 A  C. sakazakii 58 220108/52 Marinated meat 3305373 98.40 1 A  C. sakazakii 59 280108/V1 Dust 3305173 51.10 2 A  C. sakazakii 60 050208/13 Black pepper 3305173 51.10 2 A  C. sakazakii 61 050208/14 Marinated meat 3305373 98.40 1 A  C. sakazakii 62 190208/13 Marinated meat 3305373 98.40 1 A  C. sakazakii 63 120308/17 Flavoured meat 3305373 98.40 1 A  C. sakazakii 64 240608/03 Black pepper 3305373 98.40 1 A  C. sakazakii 65 240608/06 Black pepper 3305373 98.40 1 A  C. sakazakii 66 290708/08 Dry vegetable 3305373 98.40 1 A  C. sakazakii 67 290708/11 Dry vegetable 3305373 98.40 1 A  C. sakazakii 68 120808/27 Pastries 3305373 98.40 1 A  C. sakazakii 69 130808/31 Marinated meat 3305173 51.10 2 A  C. sakazakii 70 130808/32 Flavoured meat 3305373 98.40 1 A  C. sakazakii 71 130808/33 Marinated meat 3305373 98.40 1 A  C. sakazakii 72 270808/17 Marinated meat 3305373 98.40 1 A  C. sakazakii 73 100908/15 Pork swab 3305373 98.40 1 A  C. sakazakii 74 LMG 23826 a Human 3305173 51.10 9 B  C. malonaticus 75 121206/29 Black pepper 3305373 98.40 5 B  C. malonaticus 76 201206/21 a Chocolate 3305373 98.40 5 B  C. malonaticus 77 150507/66 a Nut chocolate 3325373 98.40 5 B  C. malonaticus 78 150507/70 Milk powder 3325373 98.40 5 B  C. malonaticus 79 161007/29 Meat product 3305373 98.40 5 B  C. malonaticus 80 161007/35 a Meat product 3305373 98.40 5 B  C. malonaticus 81 290108/11 a Marinated meat 3305373 98.40 5 B  C. malonaticus 82 120808/24 a Pastries 3305373 98.40 5 B  C. malonaticus 83 260808/02 Instant soup 3305373 98.40 5 B  C. malonaticus 84 LMG 23827 a Human 3305373 98.40 16 C  C. turicensis 85 290708/07 a Dry vegetable 3305373 98.40 16 C  C. turicensis 86 260808/05 Dry vegetable 3305373 98.40 16 C  C. turicensis 87 LMG 23823 a Milk powder 3345373 99.90 12 D  C. dublinensis 88 LMG 23824 a Water 3345173 99.70 10 D  C. dublinensis 89 LMG 23825 a Milk powder 3345373 99.90 6 D  C. dublinensis 90 CCM 3461 a Human 3345173 99.70 10 D  C. dublinensis Antonie van Leeuwenhoek (2011) 99:257–269 261  1 3
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