07.QXD 9-03-2007 15:08 Pagina 39 Annals of Microbiology, 57 (1) 39-47 (2007) Characterisation of Pseudomonas spp. isolated from foods Laura FRANZETTI*, Mauro SCARPELLINI Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, sezione Microbiologia Agraria Alimentare Ecologica, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Received 30 June 2006 / Accepted 27 December 2006 Abstract - Putative Pseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters: Pseudomonas fluorescens (cluster I), Pseudomonas fragi (cluster II and V) Pseudomonas migulae (cluster III), Pseudomonas aeruginosa (cluster IV) and Pseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especially Pseudomonas fluorescens. Instead Pseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities. Key words: Pseudomonas fluorescens, enzymatic activity, ITS1. INTRODUCTION the most frequently found species, however the species dis- tribution within the food ecosystem remains relatively The genus Pseudomonas is the most heterogeneous and unknown (Arnaut-Rollier et al., 1999). The principal micro- ecologically significant group of known bacteria, and bial population of many vegetables in the field consists of includes Gram-negative motile aerobic rods that are wide- species of the genus Pseudomonas, especially the fluores- spread throughout nature and characterised by elevated cent forms. During storage and processing their numbers metabolic versatility, thanks to the presence of a complex increase, and in Minimally Processed Vegetables (MPV) they enzymatic system. The nutritional requirements of play an important role in the phenomenon of browning Pseudomonas spp. are very simple, and the genus is found because of their pectinolytic activity (Ngyen-The and Carlin, in natural habitats like soil, fresh water, marine environ- 1994; Riva et al., 2001). The most representative species ments etc., but it has also been isolated from clinical instru- are Pseudomonas fluorescens, Pseudomonas putida, ments, aseptic solutions, cosmetics and medical products. Pseudomonas chicorii and Pseudomonas maltophilia. Some species are important medically because they are Fish products serve as a good growth substrate for the considered opportunistic pathogens for humans and animals Pseudomonas genus, especially under aerobic iced storage. while others, like phytopathogens, are very important in the Indeed, the genus is considered a major producer of the agricultural sector (Ridgway and Safarik, 1990; Palleroni, volatile compounds responsible for off-flavour compounds 1991a, 1993). The identification of the species of this genus (aldehydes, ketones and esters) (Gram and Huss, 1996; was first described by Migula in 1894 (Palleroni, 1984); Franzetti et al., 2001) and Pseudomonas fragi appears the however, for a long time there were many gaps. Palleroni et most to blame for fructy-odor production, a consequence of al. (1973) began a complex study on the genus and recog- amino acid degradation (Molin and Ternstrom, 1986; Miller nized 5 rRNA similarity groups. Today, only rRNA similarity et al., 1993; Tryfinopoulou et al., 2002; Garcia-Lopez et al., group 1 is considered the “true” Pseudomonas or Pseudomonas “sensu stricto”, (Moore et al., 1996) while dif- 2004). ferent designations have been proposed for the members of Pseudomonas spp. also plays an important role in milk other groups (Stanier et al., 1966; Johnson and Palleroni, spoilage. During the storage of raw milk they produce many 1989; Palleroni, 1991b; Kozo, 1995). thermo-tolerant lipolytic and proteolytic enzymes that Most members of group 1 are psychrotrophic, can be flu- reduce both the quality and shelf life of processed milk orescent or non-fluorescent, and have long been known to (Wiedmann et al., 2000; Dogan and Boor, 2003). be responsible for chilled food spoilage. Pseudomonas fluo- Thus, given the great interest in the genus rescens, Pseudomonas putida, and Pseudomonas fragi are Pseudomonas, and its primary role in the processes of food- stuff spoilage, the aim of this work was to classify, and to evaluate, the enzymatic activities of a large number of iso- * Corresponding author. Phone: +39-0250316734; lates, previously assigned to the Pseudomonas genus, com- Fax: +39-0250316694; E-mail: [email protected] ing from different foods. 07.QXD 9-03-2007 15:08 Pagina 40 40 L. Franzetti and M. Scarpellini MATERIALS AND METHODS AGAGTTTGATCCTGGCTCAG-3’; 16SR 5’-CTACGGCTAC- CTTGTTACGA-3’) and the following thermal profile: 2 min at Bacterial strains. A total of 102 isolates of putative 94 °C; 5 cycles consisting of 94 °C for 45 s, 55 °C for 1 min, Pseudomonas spp. were examined for phenotypic and geno- 72 °C for 2 min; 35 cycles consisting of 92 °C for 45 s, 60 typic characteristics. The strains, isolated since 1996 to °C for 45 s, 72 °C for 2 min; final extension of 72 °C for 2 2005 and coming from different foods, were taken from the min; and final cooling at 4 °C. Amplification specific for collection of freeze-dried cultures kept in the food-microbi- Pseudomonas DNA was performed using the primer set ology laboratory of DISTAM, Milan. The origin of the isolat- 16SF-PSMG (5’-CCTTCCTCCCAACTT- 3’) (Johnsen et al., ed strains was: 22 from minimally processed vegetables, 5 1999) and the following thermal profile: 6 min at 94 °C; 35 from pasteurised milk, 2 from fresh cheese, 16 from ground cycles consisting of 92 °C for 30s, 52 of 5 °C for 30 s, each raw beef, 11 from uncooked chicken hamburger and 46 cycle being followed by 1 min at 68 °C; on completion of all from sushi packed under modified atmosphere. The isolates the cycles a further 6 min at 68 °C, and final cooling at 4 were maintained on TSA slopes at 5 °C and sub-cultured °C. DNA 16S-23S intergenic spacer (ITS1) region amplifica- every month. All isolates are listed in Table 1. tion was performed using the primer set 16F945 and 23R458 (Lane et al., 1985) (16F945 5’-GGGCCCGCA- References strains. The study used the following refer- CAAGCGGTGG-3’; 23R458 5’-CTTTCCCTCACGGTAC-3’) and ence strains as controls: Pseudomonas fluorescens biotype the following thermal profile: 5 min at 94 °C; 30 cycles con- A ATCC 17555, Pseudomonas fluorescens biotype B ATCC sisting of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min; 17482, Pseudomonas fluorescens biotype C ATCC 17559, final extension of 72 °C for 2 min. Pseudomonas fluorescens biovar 3 DSMZ 50124, 16S rRNA gene partial region (482-521 bp; 1311-1351 Pseudomonas fluorescens biotype G ATCC 17518, bp) specific for Pseudomonas fluorescens amplification was Pseudomonas aeruginosa DSMZ 50071T, Pseudomonas performed using the primer set 16SPSEfluF and 16SPSER agarici DSMZ 11810T, Pseudomonas alcaligenes DSMZ (16SPSEfluF 5’-TGCATTCAAAACTGACTG-3’; 16SPSER 50342T, Pseudomonas asplenii DSMZ 50254, Pseudomonas 5’AATCACACCGTGGTAACCG-3’) (Scarpellini et al., 2004). chichorii DSM 50259T, Pseudomonas chlororaphis DSMZ Following amplification, 7 µl of each amplificate was 50083T, Pseudomonas fragi DSMZ 3456T, Pseudomonas analysed by electrophoresis at 100 Volt (1% agarose gel, mendocina DSMZ 50017T, Pseudomonas putida DSM 291T, 0.2 µg of ethidium bromide ml-1) in TAE buffer. The ampli- Pseudomonas syringae DSMZ 10604T, Pseudomonas fication was performed in a DNA thermal cycle (Biometra T stutzeri DSM 51909T, Pseudomonas viridiflava DSMZ gradient, Germany). 11124.T The strains were maintained on TSA slopes at 5 °C and sub-cultured every month. Restriction analysis of the 16S rRNA gene and spacer (ITS1). Restriction digestion of each amplified 16S rRNA Morphological, physiological and biochemical tests. gene was carried out for 16 h at 30 °C in 25 ml reaction Each isolate was tested for morphology, motility and Gram mixture containing 15 µl of template DNA, 2.5 µl of 10X PCR stain by phase contrast microscopy. The following physio- reaction buffer, 18.75 U of one restriction enzyme, either logical tests were performed: fluorescent and phenazine HaeIII or VspI (Amersham Pharmacia Biotech). Restriction pigment production (King et al., 1954), accumulation of digestion of each amplified ITS1 was carried out for 16 h at poly-b-hydroxybutyrate (Palleroni and Doudoroff, 1972) 65 °C in 25 µl reaction mixture containing 15 ml of template levans production (Lelliot et al., 1966), growth at 4, 10 and DNA, 2.5 µl of 10X PCR reaction buffer, 18.75 U of Taq I 41 °C and oxidative or fermentative acid production (Hugh (Amersham Pharmacia Biotech) (Guasp et al., 2000). The and Leifson, 1953), API 20NE strips (Bio Mérieux, Marcy restriction digestions were then analysed by agarose elec- l’Etoile, France) were used for a preliminary biochemical trophoresis (3% w/v), containing 0.2 µg of ethidium bro- characterisation of the strains and the species were identi- mide ml-1, TAE buffer (Guasp et al., 2000). fied using the API database. The enzymatic activities tested were: lipase production (Sierra, 1957), pectinolytic activity RFLP cluster analysis of ITS1. A computer similarities (Sand et al., 1972), starch hydrolysis (Stanier et al., 1966), analysis was estimated by means of the jaccard coefficient lecithinase production (Nutrient Agar plates plus 5% v/v (Sneath and Sokal, 1973) and clustering of strains was egg-yolk emulsion) and proteolytic enzyme production based on unweighted pair group method with arithmetic (Nutrient Agar plates containing 10% skim milk powder). average (UPGMA). The NTSYS-PC computer program (ver- sion 1.30) (Rohlf, 1987) was used in the data analysis. DNA extraction and PCR amplification. Template DNA was prepared by boiling 200 µl of bacterial suspension in Total sequencing of the16S rRNA gene. After amplifica- MilliQ (OD600 = 0.6) in safe-lock Eppendorf tubes for 10 tion of the 16S rRNA gene from extracted DNA, the PCR min.