Italian Journal of Food Safety 2016; volume 5:5620

American economic resource of the coastal communities of North America, with more than 130,000 tons Correspondence: Cristian Bernardi, Department (Homarus americanus) of alive animals commercialised worldwide in of Health, Animal Science and Food Safety, not surviving during air 2006 and producing nearly 545 million of euros University of Milan, via G. Celoria 10, 20133 transport: evaluation of profits. Due to the decrease of the European Milan, Italy. Homarus gammarus catching figures, Tel: +39.02.50317855 - Fax: +39.02.50317870. E-mail: [email protected] of microbial spoilage American lobsters are imported by several European countries, especially Mediterranean Erica Tirloni, Simone Stella, Acknowledgements: the authors would like to ones: in Italy about 4387 tons are annually thank METRO Italia SpA for providing the Mario Gennari, Fabio Colombo, introduced for an economic value of 44 million American lobsters. Professor Patrizia Cattaneo Cristian Bernardi of euros (Barrento et al., 2009; FAO, 2006). should also be acknowledged for her careful revi- Department of Health, Animal Science American lobsters are mainly commercialised sion of the manuscript. alive and cooked before consumption. After the and Food Safety, University of Milan, Key words: Dead American lobsters; Shipping; capture and during the air transport, they are Milan, Italy Food spoilage; Microbial population. submitted to several stresses like air exposure, changes of the natural environments in terms Contributions: ET and SS were involved in micro- of physical and chemical parameters such as biological analyses and writing of the paper. FC Abstract water salinity and temperature, hypoxia, fast- was involved in biomolecular analyses. MG was ing, interactions with other conspecifics and involved in phenotypical tests. CB was involved in human handling. These stressors can affect biomolecular analyses, reference search and planning of the trial. Eighteen American lobsters (Homarus their health, reducing their quality, and in americanus), dead during air transport, were some cases could be lethal. Opposite opinions analysed in order to evaluate the microbial Funding: the study was funded by the University are still existing about the edibility of lobsters of Milan, Piano di Sostegno alla Ricerca 2014, population of meat, gills and gut: no specific dead during air shipping: some sellers reject to Linea B. studies have ever been conducted so far on the sale the dead subjects, some others use these microbiological quality of American lobsters’ ones for cooked products, others sell these ani- Conflict of interest: the authors declare no poten- meats in terms of spoilage microbiota. The mals depreciated. At the moment there are no tial conflict of interest. meat samples showed very limited total viable available studies about the hygienic quality of Received for publication: 3 November 2015. counts, in almost all the cases below the level the living and dead animals, in terms of pres- of 6 Log CFU/g, while higher loads were found, Revision received: 25 January 2016. ence of potential pathogenic and of Accepted for publication: 26 January 2016. as expected, in gut and gills, the most probable microbial populations. However, we could sup- source of contamination. These data could jus- pose that the loads and the typology of micro- This work is licensed under a Creative Commons tify the possibility to commercialise these not- biota of lobsters reflect a combination of fac- Attribution-NonCommercial 4.0 International surviving subjects, without quality concerns tors including the environment where they are License (CC BY-NC 4.0). for the consumers. Most of the isolates result- captured, their feeding and living practices, ©Copyright E. Tirloni et al., 2016 ed to be clustered with type strains of the season of capture as well as the quality and spp. (43.1%) and Licensee PAGEPress, Italy temperature of the waters where they lived, as Italian Journal of Food Safety 2016; 5:5620 Photobacterium spp. (24.1%), and in particular already demonstrated for other crustaceans doi:10.4081/ijfs.2016.5620 to related to the natural marine envi- and for finfish (Liston, 1980; Shewan, 1977). ronment. The distribution of the genera After death, once the immune response fails to showed a marked inhomogeneity among the function and the muscular structure declines tion of the meat of American lobsters not sur- samples. The majority of the isolates identified due to microbial activity, the alteration begins viving the air shipping from the harvest (USA) resulted to possess proteolytic (69.3%) and quickly: generally this situation does not to a seafood distribution platform of an inter- lipolytic ability (75.5%), suggesting their expose consumer to microbiological risks as potential spoilage ability. The maintanance of these animals are mainly consumed after national wholesaler, located in Northern Italy, good hygienical practices, especially during cooking. However, the increasing demand for and the identification of the most representa- the production of ready-to-eat lobsters-based ready-to-eat raw or cooked crustaceans and of tive bacterial populations in order to establish products, and a proper storage could limit the fresh lobster tails could enhance the risk posed the major genera/species involved with micro- possible replication of these microorganisms. by these products, if subjected to improper bial spoilage of these products. We also evalu- storage or poor hygienic practices during pro- ated the microbial loads of gut and gills as pos- duction and commercialisation. sible sources of contamination. Swartzentruber et al. (1980) found high total Introduction bacterial count values [more than 5 Log colony forming unit (CFU)/g] in 51.9% of frozen lob- The 90% of fish international market is ster tails samples, even if very low levels of col- Materials and Methods based on processed products, but live crus- iforms and E. coli were detected. Although, as taceans, especially clawed and spiny lobsters, already mentioned, no studies till now were Microbiological analyses crabs, marine and freshwater and conducted on lobster meat bacterial popula- For this study, eighteen lobsters arrived freshwater crayfish, are really appreciated for tion, generally the microbiota of crustaceans dead after air transport lasted about 24 h from their high nutritional and commercial impor- captured in temperate waters includes harvest (USA) to the seafood distribution plat- tance (Fotedar and Evans, 2011). The Pseudomonas spp., Shewanella putrefaciens form, were collected. The subjects were trans- American lobster, Homarus americanus, is one and members of the group Acinetobacter- ported at refrigeration temperatures in card- of the most important commercial species (Gram and Huss, 1996). board boxes containing ice gel packs where marketed internationally and one of the major Aim of our study was the microbial evalua- they were maintained separately thanks to

[Italian Journal of Food Safety 2016; 5:5620] [page 75] Article cardboard dividers. The dead subjects were Lipolytic activity was assessed on Sierra’s transported under refrigerated conditions to agar containing Tween 80 (Sierra, 1957). The Results and Discussion the laboratory where they were analysed plates were incubated at 20°C for a week; pos- immediately. Aseptically, gills, meat and intes- itive strains showed a visible precipitation A total of 18 lobsters dead during shipping tine were withdrawn and submitted to microbi- around the colonies. were analysed: meat, gills (as the main access ological analyses: 5-10 g of each sample were All the isolates were screened for their abil- point for bacteria) and gut bacterial contami- diluted 1:10 in saline diluent (NaCl 10 g/L, ity to produce H2S. The medium used was Iron nations were screened. The Food and Drug

MgSO4·7H2O 1.53 g/L, MgCl2·6H2O 1.28 g/L, KCl agar 1 (Gram et al., 1987); each plate was pre- Administration (FDA, 2013), which has issued 0.19 g/L, tryptone 1 g/L) and serial 10-fold dilu- pared with 40 mL of agar stab inoculated. H2S guidelines in order to define the microbiologi- tions were prepared. Total viable count (TVC) production was indicated by the formation of a cal quality of some food categories, indicates was enumerated onto modified Tryptose agar black precipitate of FeS. To enable the growth in 6 Log CFU/g the microbiological limit for as described by Bernardi et al. (2015), incubat- of halophilic microorganisms, the original crustaceans raw meat. There are no specific ed at 20°C for 72 h. The number of composition of some media (Iron Agar 1) were studies about the microbiological quality of Enterobacteriaceae was determined according added of the following final salt concentra- American lobsters’ meat; studies are available with ISO 21528-2:2004 method (ISO, 2004). tions: NaCl 10 g/L, MgSO4·7H2O 1.53 g/L, on frozen crabs meat (ICMSF, 1998), indicat- Colonies of fluorescent pseudomonads were MgCl2·6H2O 1.28 g/L, KCl 0.19 g/L. ing that counts below 5 Log CFU/g can be con- observed on plates of King’s B agar (King et al., The ability to growth at 5, 30, 37 and 42°C sidered satisfactory, while those comprised 1954) in ultraviolet light, after growth for 1-3 was tested isolating each of the strain onto between 5 and 6 Log CFU/g should be consid- days at 20°C. Bioluminescent bacteria were plates of modified Tryptose agar. Moreover the ered fairly satisfactory. Moreover, for fish prod- enumerated onto glycerol based marine agar ability to grow onto modified tryptose agar ucts, the threshold value used to identify the (GMA) (Makemson et al., 1992); plates were including different salt concentration (0 and end of the shelf-life is often 6 Log CFU/g 0.5%) was evaluated preparing the plates with incubated at 20°C for 72 h and observed daily (Olafsdottir et al., 2005), even if the total bac- the different percentages of salt included. in dark. Finally H2S producing microorganisms terial count at the point of rejection can be were enumerated onto Iron Agar containing Sequencing of 16s rRNA gene around 7-8 Log CFU/g (Espe et al., 2004). sodium thiosulphate (IA1 following Gram et Isolates were then identified by 16s rRNA Generally, the values obtained from our raw al., 1987) using the pour plate technique. gene sequencing (Eurofins, Berlin, Germany) meat samples were characterised by limited Plates were incubated at 20°C for 72 h and using the standard forward primers CC-CD as loads: the average was below 5 Log CFU/g, con- observed for black colonies. described by Rudi et al. (1997). The sequences firming a good hygienic quality, while only two Phenotypical tests were analysed and compared to BIBI and samples resulted to exceed the 6 Log level (6.21 and 6.38 Log CFU/g) (Table 1). As expect- From the TVC plates of meat samples, a NCBI-Blast (http://blast.ncbi.nlm.nih. ed, gills and guts were characterised by higher number of five colonies for each of 12 subjects gov/Blast.cgi) databases and the closest type loads: in the intestine this is due to the pres- (identified with code from A to N) were ran- strains relatives based on partial 16S rRNA ence of a great amount of bacteria which con- domly picked and subcultured onto modified gene sequences were determined. In order to stitute the natural microbiota, while the gills Tryptose agar. The total of 60 isolates were verify if the number of identified taxa was rep- were identified as the main point of access of afterwards submitted to the tests as described resentative of the whole microbial species the bacteria from the outside due to the pecu- below. present in the ecological niche of American liar anatomical structure that allows gaseous The following reactions and biochemical lobsters meat, the total number of bacterial tests were used for the characterisation of the species and the percentage detected were esti- exchanges. These loads, in any case, do not isolates. Gram-reaction was tested by the KOH mated by method described by Chao et al. represent a hygienic problem as the product is method (Gregersen, 1978). Phase contrast (2009) for incidence data. generally consumed after a thermal treatment. microscopy was used for the determination of However, in order to avoid the cross contami- shape and motility. Cytochrome-oxidase was tested with the method described by Kovacs

(1956) and catalase formation with 3% H2O2. The carbohydrate metabolism was observed Table 1. Mean bacterial counts, number of countable samples and number of samples in tubes of Hugh and Leifson medium (Hugh under the 6 Log limit of meat, gills and gut of American lobsters. and Leifson, 1953). Tubes were stab inoculated Mean±SD Detection limit of 6 Log level (after boiling to avoid oxygen) and incubated (no. of countable (no. of samples at 20°C for a week. Fermentative bacteria pro- samples) under the limit) duced acid from glucose, changing the color of the indicator from green to yellow, in all the TVC 20°C Meat 4.5±0.9 (n=16) <2 (n=2) Gills 7.1±0.8 (n=12) <2 (n=0) volume of the medium. Oxidative bacteria Gut 6.5±1.3 (n=16) <2 (n=0) grew only on the surface of the medium, and the eventual change of color (blue due to Bioluminescent bacteria Meat 3.1±0.9 (n=7) <2 (n=8) Gills 4.4±1.6 (n=10) <2 (n=2) alcalinisation or yellow for acidification) was Gut 4.3±1.5 (n=8) <2 (n=5) located within 2-3 cm from the surface. Proteolytic activity was detected by gelatin H2S producing bacteria Meat 2.4±1.4 (n=6) <1 (n=9) hydrolysis on Frazier’s agar plates (Frazier, Gills 4.0±1.4 (n=7) <1 (n=3) 1926). The inoculated plates were incubated at Gut 3.3±1.4 (n=8) <1 (n=5) 20°C for a week, than flooded with Frazier’s Enterobacteriaceae Meat (n=0) <2 (n=15) reactive. Gelatinase activity was indicated by Gills 3.6±1.3 (n=3) <2 (n=9) formation of a clear zone around the positive Gut 3.7±2.0 (n=3) <2 (n=10) colonies. SD, standard deviation; TVC, total viable count.

[page 76] [Italian Journal of Food Safety 2016; 5:5620] Article nation between raw products during the prepa- Table 2. Biomolecular identification of bacterial isolates from American lobsters meat and ration, the good manufacturing and hygienic similarity rate. practices should be always applied. Subject code Isolate number Closest relative in database and similarity rate Concerning the other parameters evaluated, A 1 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) all the counts of meat samples resulted low A 2 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) (average values of bioluminescent and H S 2 A 3 Psychrobacter submarinus (99%) producing bacteria were always below 4 Log A 4 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) CFU/g). Considering H2S producing bacteria, typical spoiler of fish products, although the A 5 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) loads detected in meat samples do not reach B 6 Photobacterium kishitani/piscicola (99%) high levels, they should be carefully consid- B 7 Photobacterium kishitani (99%) ered, due to their potential replication during B 8 Photobacterium kishitani (99%) the incidental further storage at low tempera- B 9 Photobacterium kishitani/aquimaris (99%) tures. For all the parameters, higher values B 10 rumoiensis (98%) were detected in gills and gut. Considering C 11 Psychrobacter pulmonis (99%) Enterobacteriaceae and fluorescent C 12 Psychrobacter pulmonis (100%) pseudomonads, all the samples were below the C 13 Pseudoalteromonas aliena (99%) detection limit (2 Log CFU/g). The absence of C 15 Vibrio rumoiensis/litoralis (98%) detectable counts of fluorescent pseudomon- D 16 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) ads was unexpected, as these microorganisms D 17 Vibrio lentus (100%) are common contaminants of marine fishes D 18 Pseudoalteromonas marina (98%) (Huss, 1995). D 19 Pseudoalteromonas translucida (99%) In order to understand the ecology of the D 20 Psychrobacter piscatorii/nivimaris (99%) strains grown on the plates of the total viable E 21 Photobacterium frigidiphilum (99%) count of meat samples, some phenotypic traits E 22 Photobacterium frigidiphilum (99%) were considered: the 43.1% resulted to ferment E 23 Photobacterium frigidiphilum (99%) glucose while the remaining 56.9% to be E 24 Photobacterium frigidiphilum (99%) oxidative. Considering the ability to grow at E 25 Photobacterium frigidiphilum (99%) different temperatures, the psychrotrophic F 26 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) nature of the microbiota was evidenced, even F 27 Pseudomonas arctica/Alteromonas fuliginea (99%) if the ability to adapt to higher temperatures F 28 Psychromonas arctica (99%) was also observed. In fact, all the isolates grew F 29 Psychromonas arctica (99%) at refrigerated temperatures (5°C), the 46.6% F 30 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (99%) of the isolates was able to grow at 30°C while only the 3.4% resulted to be able to grow at G 31 Psychromonas arctica (99%) 37°C and none of the isolates was able to grow G 32 Psychromonas arctica (99%) at 42°C. Taking in account the salt require- G 33 Pseudoalteromonas marina (98%) ment, the 82.8% of isolates resulted to be able G 34 Polaribacter sejongensis/butkevichii (98%) to grow at 0.5% of salinity, while only the 24.1% G 35 Psychromonas arctica (99%) at 0%, evidencing that the main lobster bacte- H 36 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) rial population is composed by halotolerant H 37 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) and halophilic microorganisms. From the 60 H 38 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) isolates randomly picked from the plates of H 39 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) meat samples, 2 were lost due to their short H 40 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) surviving in vitro. All the isolates were Gram I 41 Alivibrio logei/salmonicida (100%) Negative (Table 2). I 42 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) The isolates clustered mostly (43.1%) with I 43 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) type strains from Pseudoalteromonas spp.: this I 44 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) is one of the largest genera within the I 45 Pseudoalteromonas traslucida (99%) and currently comprises L 46 Pseudomonas sabulinigri (98%) more than 30 species (Ivanova et al., 2004). L 47 Psychromonas arctica (99%) These microorganisms are characterised by a L 49 Shewanella vesiculosa/livingstonensis (98%) widespread distribution across many marine L 50 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) habitats (e.g., seawater, rocks, macroalgae and M 51 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) marine animals) and are able to survive in M 52 Shewanella arctica/frigidimarina (100%) poor nutrient substrates producing a varied M 53 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) range of metabolites and enzymes (Ivanova et M 54 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) al., 2003). The phylogenetic structure of this M 55 Pseudoalteromonas paragorgicola/elyakovi/distincta/nigrifaciens (100%) group indicates that the non-pigmented N 56 Photobacterium kishitani/piscicola (99%) species are strictly related among them and constitute the major cluster of the genus. In N 57 Photobacterium aquimaris (98%) our case, 25 isolates were identified as N 58 Photobacterium kishitani/piscicola (99%) Pseudoalteromonas spp.: 20 isolates (80%) N 59 Photobacterium kishitani/piscicola (99%) resulted to be closely related to the species Ps. N 60 Photobacterium kishitani/piscicola (99%)

[Italian Journal of Food Safety 2016; 5:5620] [page 77] Article paragorgicola, elyakovi, distincta and nigrifa- from samples of the Southern Ocean environ- spp.: one as Pseudomonas sabulinigri, firstly ciens. Our sequences did not allow the distinc- ment (Heuchert et al., 2004). As shown by isolated from black sand collected from Korea tion of one of these 4 species due to the high Yumoto et al. (2010), the two species P. pisca- (Kim et al., 2009) and one as Pseudomonas similarity existing among them. Two isolates torii and P. nivimaris have a deep genetic relat- arctica/Alteromonas fuliginea. resulted to be similar to Ps. marina, two as Ps. edness. Three isolates were identified as The remaining two isolates were identified as translucida and one as Ps. aliena, all often iso- Vibrio spp. (5.2%): one resulted to be related to Alivibrio logei/salmonicida, two psychrophilic lated from marine environment (Nam et al., Vibrio lentus, firstly isolated from species whose substantial similarities were 2007; Ivanova et al., 2002, 2004). Mediterranean (Macian et al., 2001), highlighted by multigenic analysis (Ast et al., Fourteen colonies (24.1%) were identified and the other to Vibrio rumoiensis, firstly iso- 2009), and as Polaribacter sejongensis/butke- as Photobacterium spp., genus that comprises lated from drain pool of a fish product process- vichii firstly isolated from Antarctic soil and 19 species, five of those containing luminous ing plant (Yumoto et al., 2013). The third iso- from sea water collected in the Sea of Japan strains (Ph. phosphoreum, Ph. kishitani, Ph. late was close to V. rumoiensis/litoralis: these (Kim et al., 2013; Nedashkovskaya et al., 2005). angustum, Ph. ganghwense, Ph. leiognathi): all two species are considered strictly related, as Keeping into account only unique identifi- these bacteria are commonly present in observed by Nam et al. (2007). Some species of cations, total number of bacterial species was marine environments and occur in seawater, Vibrio (Vibrio vulnificus and V. parahaemolyti- 68: the 13 unique species observed are nearly surfaces and gut of marine animals, marine cus in particular) are primary causes of human 19% of total number of species. Nevertheless, sediments and saline lake water, as well as in mortality and illness due to gastroenteritis or considering ambiguous identifications, an the organs of fishes (Baumann and Baumann, septicemia through consumption of raw or algorithm was build up and performed in SAS 1984; Dalgaard, 1995; Urbanczyk et al., 2011). undercooked seafood or from wound infections software (SAS Institute, Cary, NC, USA) by Five of the isolates resulted to be related to Ph. (Constantin de Magny et al. 2009). The major- which, the estimates were calculated for frigidiphilum, a psychrophilic and weakly ity of V. vulnificus and V. parahaemolyticus ill- 100,000 random possible species compositions halophilic strain, firstly isolated and identified nesses are often related with shellfish con- of a sample. According with this simulation, from deep-sea sediments of Edison Seamount sumption; nevertheless, these bacteria can the highest value found for number of bacteri- (Seo et al., 2005). Seven other isolates showed also be found in fish, sediment, plankton and al species was 53, while the mean number was to be close to Ph. kishitani: for two isolates the marine water: in our study we did not focus on 37. Number of total bacterial species colonis- species was confirmed with both the align- the presence and isolation of pathogenic bac- ing lobster meat calculated according only ment tools while for five of them the species teria, such as Vibrio spp., thus the colonies unique identifications should be considered as indicated in Blast algorithm differed and identified were isolated from a non-selective an over-estimation: as a consequence, values resulted as Ph. piscicola, a recent new one medium used for the enumeration of total coming from simulation are better estimates (Figge et al., 2014), not yet included in the viable count, and not from a specific medium of such species richness. LeBibi data bank. One strain was related to Ph. for the isolation of these bacteria. The results of our identifications partially aquimaris and one resulted to be similar with Two strains were identified as Shewanella agree with those obtained by Gornik et al. the same score to both the species Ph. kishi- spp.: one resulted to be similar to the species (2011) in Norway lobster (Nephrops norvegi- tani and Ph. aquimaris: according to S. vesiculosa, firstly isolated from marine sedi- cus) tail meat. In fact most of the microbial Yoshizawa et al. (2009), who firstly identified ments collected at Deception Island, and S. liv- spoilage population found was composed by the latter species, these two are recognised to ingstonensis, firstly isolated from Antarctic the same genera Vibrio spp., Photobacterium be very closely related from a genetic point of coastal marine environments (Bozal et al., spp., Pseudoalteromonas spp., Shewanella spp. view. All the six isolates recognised as 2002; 2009). The other strain was related to and Psychrobacter spp., but the relative fre- Psychromonas spp. (10.3% of the whole) Shewanella arctica/frigidimarina, firstly isolat- quencies were quite different. In particular, resulted to be close to Ps. arctica, firstly identi- ed respectively from Arctic marine sediment in the most prevalent genus in American lobsters fied in 2002: these cold-adapted microorgan- a fjord branch in Norway and from sea ice from was Pseudoalteromonas spp., while only few isms could be isolated from permanently but Antarctic meromictic lakes (Bowman et al., Vibrio spp. were found, while in Norway lob- also temporarily cold locations (e.g., oceans, 1997a; Kim et al., 2012). sters, Vibrio spp. represented more than half of polar regions, high mountains, deep lakes), Two strains were identified as Pseudomonas the isolates and only 14.6% belonged to the and are often associated with plants and fresh and cold blooded fishes and crustaceans (Groudieva et al., 2003). Four isolates clustered with the genus Psychrobacter spp. (6.9% of the whole): these Table 3. Lipolytic and proteolytic abilities of the fifty-eight isolates from American lob- microorganisms are usually halotolerant and sters meat. psychrophilic and are linked to several differ- Lipolytic activity (%) Proteolytic activity (%) ent substrates like fish skin, gills and gut, sea water but also lamb carcasses (Bowman et al., Total of the colonies identified 75.5 (46/58) 69.3 (38/58) 1997b; Gonzalez et al., 2000; Prieto et al., Pseudoalteromonas spp. 100 (24/24) 100 (24/24) 1992). Two of these isolates resulted to be Photobacterium spp. 50.0 (7/14) 35.7 (5/14) related to P. pulmonis, the only species not related to marine environment as firstly isolat- Psychromonas spp. 83.3 (5/6) 16.7 (1/6) ed and identified in 2002 from a pure culture Psychrobacter spp. 50.0 (2/4) 0 (0/4) from lung clinical specimen of lambs. Another Vibrio spp. 66.7 (2/3) 33.3 (1/3) one resulted to be close to P. submarinus, first- Pseudomonas spp. 100 (2/2) 50 (1/2) ly isolated from sea water (Romanenko et al., Shewanella spp. 100 (2/2) 100 (2/2) 2002) and another one to P. piscatorii/nivi- Polaribacter spp. 0 (0/1) 0 (0/1) maris, species firstly isolated from a drain of a fish-processing plant (Yumoto et al., 2010) and Alivibrio spp. 0 (0/1) 0 (0/1)

[page 78] [Italian Journal of Food Safety 2016; 5:5620] Article genus Pseudoalteromonas. characterization of spoilage bacteria from Considering the genera identified, a clear packed fish. Int J Food Microbiol 26:319- inhomogeneity in the samples distribution 33. was observed with some of them showing a References Dalgaard P, 2000. Freshness, quality and safety strict prevalence of Pseudoalteromonas spp. in seafoods. Flair-flow Europe technical (e.g., subjects code A, D, H, I, M) and some oth- Ast J, Urbanczyk H, Dunlap PV, 2009. Multi- manual F-FE 380A ⁄ 00. Teagasc-The ers of Photobacterium spp. (B, E, N). As a gene analysis reveals previously unrecog- National Food Centre, Dublin, Ireland. result, a single subject is not reflecting the nized phylogenetic diversity in Aliivibrio. Espe M, Kiessling A, Lunestad B-T, Torrissen microbial population of all the conspecifics and Syst Appl Microbiol 32:379-86. OJ, Bencze AM, Lebensm R, 2004. Quality consequently it is quite difficult to predict the Barrento S, Marques A, Teixeira B, Vaz-pires P, of cold smoked salmon collected in one development of the spoilage microbiota in this Nunes ML, 2009. Nutritional quality of the French hypermarket during a period of 1 product. In any case, all the identified spoilage edible tissues of European lobster year. Food Sci Technol 37:627-38. microorganisms were common natural marine Homarus gammarus and American lobster FAO, 2006. The state of fisheries and aquacul- bacteria, and no important post-harvest con- Homarus americanus. J Agr Food Chem ture. Fisheries and Aquaculture tamination by not halophilic bacteria, due to 57:3645-52. Department, Food and Agriculture handling or cross contamination, was evi- Baumann P, Baumann L, 1984. Genus II. Organization, Rome, Italy. Available from: denced. All the genera, apart from Vibrio spp., Photobacterium Beijerink 1889, 401AL. In: ftp://ftp.fao.org/docrep/fao/009/a0699e/a06 are microbial spoilers of seafood (Dalgaard, Krieg NR, Holt JG, eds. Bergey’s manual of 99e.pdf 2000; Gram and Huss, 1996; Gram et al., 1987; systematic bacteriology. Williams and Figge MJ, Cleenwerck I, van Uijen A, De Vos P, Huss, 1995; Shewan, 1977). Wilkins, Baltimore, MD, USA, pp 539-45. Huys G, Robertson L, 2014. All the isolates were screened for their pro- Bernardi C, Baggiani L, Tirloni E, Stella S, Photobacterium piscicola sp. nov., isolated teolytic and lipolytic ability (Table 3), showing Colombo F, Moretti VM, Cattaneo P, 2015. from marine fish and spoiled packed cod. the wide diffusion of metabolically active Hemolymph parameters as physiological Syst Appl Microbiol 37:329-35. strains. In particular, all Pseudoalteromonas biomarkers in American lobster Homarus FDA, 2013. Revised guidelines for the assess- spp. isolates resulted to possess lipolytic abili- americanus for monitoring the effects of ment of microbiological quality of ties and also proteolytic capabilities. two commercial maintenance methods. processed food. Food and Drug Photobacterium spp., Psychrobacter spp., Fish Res 161:280-4. Administration, Silver Spring, MD, USA. Vibrio spp. and Psychromonas spp. showed bet- Bowman JP, McCammon SA, Nichols DS, Fotedar S, Evans L, 2011. Health management ter lipolytic activity than proteolytic. None of Skerratt JH, Rea SM, Nichols PD, during handling and live transport of crus- the isolates in this study showed the ability to McMeekin TA, 1997a. Shewanella gelidi- taceans: a review. J Invertebr Pathol produce H2S: this finding is in agreement with marina sp. nov. and Shewanella frigidima- 106:143-52. the data obtained from fresh Norway lobsters rina sp. nov., novel Antarctic species with Frazier WC, 1926. A method for the detection of (Gornik et al., 2011) where all the isolates the ability to produce eicosapentaenoic changes in gelatin due to bacteria. J Infect showed not to possess this capability. acid 20:53 and grow anaerobically by dis- Dis 39:302-9. similatory FeIII reduction. Int J Syst Gonzalez CJ, Santos JA, García-López M-L, Bacteriol 47:1040-7. Otero A, 2000. Psychrobacters and related Bowman JP, Nichols DS, McMeekin TA, 1997b. bacteria in freshwater fish. J Food Protect Conclusions Psychrobacter glacincola sp. nov., a halo- 3:299-418. tolerant, psychrophilic bacterium isolated Gornik SG, Albalab A, Macpherson H, Birkbeck To our knowledge, no specific studies have from Antarctic Sea ice. Syst Appl Microbiol H, Neil DM, 2011. The effect of tempera- been conducted so far about the microbiologi- 20:209-15. ture on the bacterial load and microbial cal quality of American lobsters’ meat in terms Bozal N, Montes MJ, Galbis DM, Manresa A, composition in Norway lobster Nephrops of spoilage microbial population. The genera Mercade E, 2009. Shewanella vesiculosa norvegicus tail meat during storage. J Appl isolated from meat samples, mostly charac- sp. nov., a psychrotolerant bacterium iso- Microbiol 111:582-92. terised by marine Pseudoalteromonas spp. and lated from an Antarctic coastal area. Int J Gram L, Huss HH, 1996. Microbiological Photobacterium spp., could be considered rap- Syst Evol Micr 59:336-40. spoilage of fish and fish products. Int J resentative of the microbiota of not-surviving Bozal N, Montes MJ, Tudela E, Jimenez F, Food Microbiol 33:121-37. subjects, but our data could also reflect the Guines J, 2002. Shewanella frigidimarina Gram L, Trolle G, Huss HH, 1987. Detection of microbial environment of live lobsters, trans- and Shewanella livingstonensis sp. nov. specific spoilage bacteria from fish stored ported and stored in similar conditions. The isolated from Antarctic coastal areas. Int J at low 0°C and high 20°C temperatures. evaluation of not surviving lobsters is of partic- Syst Evol Micr 52:195-205. Int J Food Microbiol 4:65-72. ular concern as they could be an optimal sub- Chao A, Colwell RK, Lin C-W, Gotelli NJ, 2009. Gregersen T, 1978. Rapid method for distinc- strate for the growth of spoilage bacteria. Our Sufficient sampling for asymptotic mini- tion of Gram-negative and Gram-positive samples showed very limited microbial loads, mum species richness estimators. Ecology bacteria. Eur J Appl Microbiol Biotech suggesting the possibility to commercialise 90:1125-33. 5:123-7. these subjects without particular quality faults Constantin de Magny G, Long W, Brown C, Groudieva T, Grote R, Antranikian G, 2003. for the consumers. The application of good Hood R, Huq A, Murtugudde R, Colwell R, Psychromonas arctica sp. nov., a novel psy- hygienical practices, especially during the pro- 2009. Predicting the distribution of Vibrio chrotolerant, biofilm-forming bacterium duction of ready-to-eat lobsters-based prod- spp. in the Chesapeake Bay: a Vibrio isolated from Spitzbergen. Int J Syst Evol ucts, and a proper ice storage could limit the cholerae case study. EcoHealth 6:378-89. Micr 53:539-45. possible replication of these microorganisms. Dalgaard P, 1995. Qualitative and quantitative Heuchert A, Glöckner FO, Amann R, Fischer U,

[Italian Journal of Food Safety 2016; 5:5620] [page 79] Article

2004. Psychrobacter nivimaris sp. nov., a 59:38-41. Environ Microb 58:2245-9. heterotrophic bacterium attached to Kim S-J, Park S-J, Oh Y-S, Lee S-A, Shin K-S, Romanenko LA, Schumann P, Rohde M, organic particles isolated from the South Roh D-H, Rhee S-K, 2012. Shewanella arc- Lysenko AM, Mikhailov VV, Stackebrandt E, Atlantic Antarctica. Syst Appl Microbiol tica sp. nov., an iron-reducing bacterium 2002. Psychrobacter submarinus sp. nov. 27:399-406. isolated from Arctic marine sediment. Int J and Psychrobacter marincola sp. nov., psy- Hugh R, Leifson E, 1953. The taxonomic signif- Syst Evol Micr 62:1128-33. chrophilic halophiles from marine envi- icance of fermentative versus oxidative King EO, Ward MK, Raney DE, 1954. Two sim- ronments. Int J Syst Evol Micr 52:1291-7. metabolism of carbohydrates by various ple media for the demonstration of Rudi K, Skulberg OM, Larsen F, Jacoksen KS, gram-negative rods. J Bacteriol 66:24-6. pyocyanin and fluorescin. J Lab Clin Med 1997. Strain classification of oxyphotobac- Huss HH, 1995. Quality and quality changes in 44:301-7. teria in clone cultures on the basis of 16S fresh fish. Food and Agriculture Kovacs N, 1956. Identification of Pseudomonas rRNA sequences from variable regions V6, Organization, Rome, Italy. pyocyanea by the oxidase reaction. Nature V7 and V8. Appl Environ Microb 63:2593-9. ICMSF, 1998. Microorganisms in Foods 6: 178:703. Seo HJ, Bae SS, Lee J-H, Kim S-J, 2005. microbial ecology of food commodities. Liston J, 1980. Microbiology in fishery science. Photobacterium frigidiphilum sp. nov., a International Commission on In: Connell JJ, ed. Advances in fishery sci- psychrophilic, lipolytic bacterium isolated Microbiological Specifications for Foods, ence and technology. Fishing News Books, from deep-sea sediments of Edison New York, NY, USA. Farnham, UK, pp 138-157. Seamount. Int J Syst Evol Micr 55:1661-6. ISO, 2004. Microbiology of food and animal Macian MC, Ludwig W, Aznar R, Grimont PAD, Shewan JM, 1977. The bacteriology of fresh feeding stuffs. Horizontal methods for the Schleifer KH, Garay E, Pujalte MJ, 2001. and spoiling fish and the biochemical detection and enumeration of Vibrio lentus sp. nov., isolated from changes induced by bacterial action. Torry Enterobacteriaceae. Part 2: colony-count Mediterranean oysters. Int J Syst Evol Micr Research Station, Torry, UK. method. ISO Norm 21528-2:2004. 51:1449-56. Sierra G, 1957. A simple method for the detec- International Organization for Makemson JC, Fulayfil N, Basson P, 1992. tion of lipolytic activity of micro-organisms Association of luminous bacteria with arti- Standardization, Geneva, Switzerland. and some observations on the influence of Ivanova EP, Bakunina Y, Nedashkovskaya OI, ficial and natural surfaces in Arabian Gulf the contact between cells and fatty sub- Gorshkova NM, Alexeeva YV, 2003. seawater. Appl Environ Microb 58:2341-3. strates. A Van Leeuw J Microb 23:15-22. Ecophysiological variabilities in ectohy- Nam Y-D, Chang H-W, Park JR, Kwon H-J, Swartzentruber A, Schwab AH, Duran AP, drolytic enzyme activities of some Quan X-Z, Park Y-H, Kim B-C, Bae JW, Wentz BA, Read Jr. RB, 1980. Pseudoalteromonas species, P. citrea, P. 2007. Vibrio litoralis sp. nov., isolated from Microbiological quality of frozen shrimp issachenkonii, and P. nigrifaciens. Curr a Yellow Sea tidal flat in Korea. Int J Syst and lobster tail in the retail market. Appl Microbiol 46:6-10. Evol Micr 57:562-5. Environ Microb 40:765-9. Ivanova EP, Gorshkova NM, Zhukova AM, Nam Y-D, Chang H-W, Park JR, Kwon H-J, Urbanczyk H, Ast JC, Dunlap PV, 2011. Zelepuga EA, Prokof’eva VV, Nicolau DV, Quan Z-X, Park YH, Lee J-S, Yoon J-H, Bae Phylogeny, genomics, and symbiosis of Christen R, 2004. Characterization of J-W, 2007. Pseudoalteromonas marina sp. Pseudoalteromonas distincta-like sea- nov., a marine bacterium isolated from Photobacterium. FEMS Microbiol Rev water isolates and description of tidal flats of the Yellow Sea, and reclassifi- 35:324-42. Pseudoalteromonas aliena sp. nov.. Int J cation of Pseudoalteromonas sagamiensis Yoshizawa S, Wada M, Kita-Tsukamoto K, Syst Evol Micr 54:1431-7. as Algicola sagamiensis comb. nov.. Int J Yokota A, Kogure K, 2009. Photobacterium Ivanova EP, Sawabe T, Alexeeva YV, Lysenko Syst Evol Micr 57:12-8. aquimaris sp. nov., a luminous marine AM, Gorshkova NM, Hayashi K, Zukova NV, Nedashkovskaya OI, Kim SB, Lysenko AM, bacterium isolated from seawater. Int J 2002. Pseudoalteromonas issachenkonii Kalinovskaya NI, Mikhailov VV, Kim IS, Syst Evol Micr 59:1438-42. sp. nov., a bacterium that degrades the Bae KS, 2005. Polaribacter butkevichii sp. Yumoto I, Hirota K, Kimoto H, Nodasaka Y, thallus of the brown alga Fucus nov., a novel marine mesophilic bacterium Matsuyama H, Yoshimune K, 2010. evanescens. Int J Syst Evol Micr 52:229-34. of the family Flavobacteriaceae. Curr Psychrobacter piscatorii sp. nov., a psy- Kim CB, Oh HW, Park D-S, Hong SG, Lee HK, Microbiol 51:408-12. chrotolerant bacterium exhibiting high Bae KS, 2013. Polaribacter sejongensis sp. Olafsdottir G, Chanie E, Westad F, Luten J, catalase activity isolated from an oxidative nov., isolated from Antarctic soil, and Kristbergsson K, 2005. Prediction of environment. Int J Syst Evol Microbiol emended descriptions of the microbial and sensory quality of cold 60:205-8. genus Polaribacter, Polaribacter butke- smoked atlantic salmon Salmo salar by Yumoto I, Ichihashi D, Iwata H, Istokovics A, vichii and Polaribacter irgensii. Int J Syst electronic nose. J Food Sci 70:563-74. Ichise N, Matsuyama H, Okuyama H, Evol Micr 63:4000-5. Prieto M, García-Armesto MR, García-López Kawasaki K, 2013. Purification and char- Kim K-H, Roh HK, Chang HK, Nam Y-D, Yoon ML, Otero A, Moreno B, 1992. Numerical acterization of a catalase from the faculta- J-H, Jeon CO, Oh H-M, Bae JW, 2009. of gram-negative, nonmotile, tively psychrophilic bacterium Vibrio Pseudomonas sabulinigri sp. nov., isolated nonfermentative bacteria isolated during rumoiensis S-1T exhibiting high catalase from black beach sand. Int J Syst Evol Micr chilled storage of lamb carcasses. Appl activity. J Bacteriol 182:1903-9.

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