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1、Bio lm formation by Vibrio parahaemolyticus on food and food contact surfaces increases with rise in temperatureNoori Han a , Md. Furkanur Rahaman Mizan a , Iqbal Kabir Jahid a , b , Sang-Do Ha a , *a School of Food Science and Technology, Chung-Ang University, 72e 1Nae-Ri, Daedeok-Myun, Anseong, Gy

2、unggido, 456e 756, South Korea bDepartment of Microbiology, Jessore Science and Technology University, Jessore, 7408, Bangladesha r t i c l e i n f oArticle history:Received 8March 2016Received in revised form 26May 2016Accepted 29May 2016Available online 30May 2016Keywords:Vibrio parahaemolyticus T

3、emperature Bio lm Crab ShrimpStainless steela b s t r a c tVibrio parahaemolyticus is recognized as a human foodborne pathogen that is mostly associated with seafood. This pathogen can form a mature bio lm on food and food contact surfaces during food pro-cessing. The present study investigated V. p

4、arahaemolyticus bio lm formation at various temperatures on shrimp, crab, and stainless steel coupons, using the bio lm formation index (BFImethod on microtiter plates. The results were also con rmed by eld emission scanning electron microscopy. Both the BFI values and cultural counts revealed that

5、V. parahaemolyticus bio lm formation was stronger at higher temperatures than at lower temperatures. Bio lm formation differed according to the growth surface type and growth temperature. It was found that higher temperatures (15e 37 C induced stronger bio lm formation whereas 4and 10 C resulted in

6、attachment of the bacterial cells as monolayers. It could be concluded that temperatures of 25e 37 C result in signi cantly stronger bio lm formation as well as exoprotease and AI-2production on food and food contact surfaces, indicating that these tem-peratures might be threatening conditions for f

7、ood safety.©2016Published by Elsevier Ltd.1. IntroductionThe gram-negative bacterium Vibrio parahaemolyticus is asso-ciated with seafood-borne illness and is recognized as a human pathogen. It is isolated from a variety of raw seafoods, particularly shell sh. The pathogen is a common cause of f

8、oodborne illnesses in many Asian countries, including China, Japan, and Korea (Su &Liu, 2007. Food-poisoning outbreaks attributed to V. parahaemolyticus occur at a high frequency in Asia. In Korea, according to a recent report, the bacterium was involved in 9e 16%of the total food-poisoning case

9、s reported. In particular, on a national scale, V. parahaemolyticus food-poisoning outbreaks occurred in 78pa-tients in Korea (MFDS, 2014. This bacterial species usually attaches to underwater surfaces and is generally isolated from a variety of raw seafoods. It is mainly associated with outbreaks r

10、elated to the consumption of shell sh (Xu, Wang, Sun, Liu, &Li, 2013. Recently, V. parahaemolyticus has been implicated in an outbreak due to the consumption of raw oysters (Newton et al., 2014.Bio lms are architecturally complex assemblies of microorgan-isms on or in biotic or abiotic surfaces

11、and interfaces, characterizedby interactions between the populations. Bio lms contain exopo-lymeric substances and survive as self-organized, three-dimen-sional structures that exhibit altered phenotypic and genotypic characters (Jahid &Ha, 2012; Mizan, Jahid, &Ha, 2015. V. parahaemolyticus

12、is known to form bio lms on seafood (Rajkowski, Fratamico, Annous, &Gunther, 2009. Cell attachment and bio lm formation have also been studied in some of the marine vibrios, including Vibrio harveyi (Karunasagar, Otta, &Karunasagar, 1996, Vibrio cholerae (Faruque et al., 2006, Vibrio vulni c

13、us (Joseph &Wright, 2004, and V. parahaemolyticus (Elexson et al., 2013. Like other bio lm-producing microorganisms, V. parahaemolyticus is capable of producing distinct types of adherence factors that enable the bacterium to adhere to the surface and initiate bio lm forma-tion (Donlan, 2002.The

14、 importance of different temperature effects on virulence factors and bio lm formation has been ascertained for different microorganisms, such as Enterococcus spp. (Jahan &Holley, 2014, Salmonella spp. (Stepanovic, Cirkovic, Ranin, &Svabi c -Vlahovi c , 2004, V. vulni cus (McDougald, Lin, Ri

15、ce, &Kjelleberg, 2006, and Listeria monocytogenes (Di Bonaventura et al., 2008. As these are environmental microorganisms, it would be very common for their survival and bio lm formation to be modulated by temperature.*Corresponding author. Ha. Contents lists available at ScienceDirectFood Contr

16、ol cate/foodcontFood Control 70(2016161e 166Quorum sensing is the population-density-dependent manner by which microorganisms communicate and coordinate with intraspecies and interspecies members by secreting signaling molecules. The importance of quorum sensing for foods has been reviewed by severa

17、l authors (Bai &Rai, 2011; Mizan et al., 2015; Skandamis &Nychas, 2012; Smith, Fratamico, &Novak, 2004. When the bacterial population reaches a speci c concentration, they secrete autoinducers (AIsinto the surrounding environment. Once a speci c concentration of AIs is reached, the molec

18、ules diffuse back into their producing bacteria and induce target genes, essentially at the stationary phase, which consequently change the behavior of the bacteria (Daniels, Vanderleyden, &Michiels, 2004. The objective of this study was to assess the bio lm formation, exoprotease and AI-2produc

19、tion of V. parahaemolyticus on stainless steel (SSand seafood (craband shrimp surfaces at different temperatures.2. Materials and methods2.1. Bacterial strains, culture conditions, and growth conditions Vibrio parahaemolyticus KCTC 2471(isolatedfrom a case of food poisoning, KCTC 2729(isolatedfrom a

20、 patient suffering from “Shirasu ”(thefry of sardine boiled and sold in a half dried state ”food poisoning, and ATCC 33844(isolatedfrom a patient with food poisoning were used as a cocktail in this study. Prior to each experiment in our laboratory, the strains were activated by trans-ferring them fr

21、om stocks stored at À80 C to thiosulfate-citrate-bile salts-sucrose (TCBSagar (Difco,Becton, Dickinson and Company, Franklin Lakes, NJ, USA and incubating them overnight at 30 C. A single colony from each plate was inoculated into 5mL of tryptic soy broth (TSB;Difco containing 2.5%NaCl and incu

22、bated over-night at 30 C in a shaking incubator (VS-8480;Vision Scienti c, Daejeon Si, South Korea at 220rpm. Subsequently, the V. parahaemolyticus cultures were centrifuged at 11,000g for 10min, washed, and resuspended in fresh LB broth (2%NaCl; Difco Labo-ratories to obtain a nal optical density a

23、t 600nm (OD600 of 1.0. These cultures were diluted as required and used in subsequent planktonic growth, bio lm formation, exoprotease, and quorum sensing assays at different temperatures of 4, 10, 15, 20, 25, 30, and 37 C. These cultures are referred to as “standardized cultures ”throughout the tex

24、t.2.2. Quantitative bio lm formation assay in microtiter plates This experiment was conducted as described previously by Jahid, Lee, Kim, and Ha (2013with some modi cation. After the cultures had been grown in 2.5%NaCl-containing TSB for 24h with shaking, the standardized cultures were diluted (1:50

25、in 2.5%NaCl-containing TSB and 100-m L aliquots were placed in each well of 96-well polystyrene microtiter plates (BectonDickinson Lab-ware; Becton, Dickinson and Company. The microtiter plates were incubated at different temperatures (viz.,4, 10, 15, 20, 25, 30, or 37 C for 72h without shaking. Aft

26、er incubation, the optical den-sity of the total bacteria in the microtiter plates was measured at a wavelength of 595nm (OD595 with a microtiter plate reader (SpectraMax 190; Molecular Devices, Sunnyvale, CA, USA. The plates were washed by submersion into a small tub of water after the bacterial cu

27、ltures had been discarded. The plates were air-dried overnight and stained with 125m L of 0.1%(w/vcrystal violet dye (CV;Sigma-Aldrich, St. Louis, MO, USA for 45min at room tem-perature, and then again dried overnight. The CV was solubilized using 125m L of 95%(v/vethanol at room temperature for 10m

28、in and the absorbance was read at 570nm using a microtiter reader. The bio lm formation index (BFIwas determined by applying theequation described by Teh, Flint, and French (2010:BFI ¼AB ÀCW GB ÀGWwhere AB is the OD 595of the CV-stained attached microorganisms, CW is the OD 595of the

29、stained blank wells containing microorganism-free medium only, GB is the OD 570of the cell growth in suspended culture, and GW is the OD 570of the blank well. The degree of bio lm formation was classi ed according to Naves et al. (2008:none (BFI<0.35, weak (0.35 BFI 0.69, moderate (0.70 BFI 1.09,

30、 and strong (BFI! 1.10.2.3. Preparation of stainless steel coupons, bio lm formation, and detachment populationAustenitic SS (Type302; Chung-Ang Scienti c Inc., Seoul, Korea coupons (2Â2Â0.1cm were processed as described by Shen et al. (2012. V. parahaemolyticus cells were centrifuged, was

31、hed, and resuspended in TSB containing 2.5%NaCl. The suspension was diluted to 1:50and inoculated into 50mL Falcon tubes containing a SS coupon that was completely submerged in 10mL of TSB. The tubes were incubated at 4, 10, 15, 20, 25, 30, or 37 C for 24h to allow bio lm formation on the SS coupons

32、. Following the incuba-tion, each SS coupon was transferred to a small Petri dish (55Â12mm containing 1mL of 0.1%peptone water (PWand then agitated by rotating it clockwise and anticlockwise, using sterile tweezers. Agitation was always performed by the same person, thus it was assumed that the

33、 same amount of pressure was applied to all coupons. The removed cells were subsequently vortexed and diluted in PW for enumeration. Cell numbers were quanti ed after incubation on TCBS agar for 24h.2.4. Preparation of inoculum for food samplesThe cultures in TSB containing 2.5%NaCl were centrifuged

34、 (11,000g for 10min at 4 C and the pellets were washed with sterile phosphate-buffered saline (PBS,pH 7.2. The pellets were resuspended in the appropriate amount of PBS to make up the same nal concentration of bacterial cells. These inocula were used to form bio lm on crab and shrimp coupons.2.5. Pr

35、eparation of shrimp and crab coupons, bio lm formation, and detachment populationCrab and shrimp were purchased from a local grocery store in Anseong, Korea. The shell sh were cut with a sterile laboratory scissor into 2Â2cm 2coupons that were then washed with sterile distilled water to remove

36、the esh. Prior to inoculation with V. parahaemolyticus , the coupons were placed in an open sterile Petri dish and subjected to ultraviolet-C treatment for 30min on each side to minimize the background ora. Each coupon was then submerged in 10mL of fresh water and the bacteria were inocu-lated at a

37、1:2500dilution. The dishes were incubated for 24h, without shaking, at different temperatures (4,10, 15, 20, 25, 30, 35, and 37 C. Following incubation and for the detachment of mi-crobial populations from the coupons, the procedures described by Jahid, Han, Srey, and Ha (2014were applied with minor

38、 modi -cations. The coupons were placed in 10mL of PW (Oxoid,UK into a sterile stomacher bag (Whirl-Pak;Nasco, Fort Atkinson, WI, USA and processed using a stomacher (BagMixer;Interscience, Saint-Nom-la-Bret eche, France at the highest speed for 2min to release the bio lm-forming bacteria from the s

39、amples. Enumeration of V. parahaemolyticus was carried out by serial dilution and spread plating onto TCBS agar. The plates were incubated at 37 C for 24hN. Han et al. /Food Control 70(2016161e 166162and colonies were counted and expressed as CFU/cm2for bio lm populations.2.6. Exoprotease assayExopr

40、otease activity was assessed using a Fluoro Protease assay kit (G-Bioscience,St. Louis, MO, USA. Cultures grown overnight were diluted (1:50with standardized culture in fresh TSB broth (with2.5%NaCl and incubated for 24h without shaking at 4, 10, 15, 20, 25, 30, 35, or 37 C. After incubation, the su

41、pernatants were collected by centrifugation at 15,000g for 10min. The supernatant (50m L from each temperature condition was added to 100m L of uorescein-isothiocyanate-conjugated substrate and incubated at room temperature for 1h. Fluorescence was measured at 485nm excitation and 530nm emission wav

42、elengths, using a uorescence microplate reader (SpectraMax Gemini EM; Molecular Devices. The data were interpreted using the trypsin standard supplied with the kit. The medium with uorescent substrate was used as the negative control.2.7. Autoinducer-2determinationProduction of the autoinducer-2(AI-

43、2protein from V. parahaemolyticus at different temperatures (4e 37 C was determined according to previous procedures described (Soni et al., 2008 with minor modi cations. V. parahaemolyticus was grown on crab coupons in a cyanobacteria BG-11fresh water solution (SigmaAldrich, with incubation at diff

44、erent temperatures as previously described, without shaking. The supernatant that contained the quorum sensing molecules was centrifuged at 15,000g for 10min. Thereafter, the supernatants from the cell-free culture were passed through 0.2-m m Tuffryn syringe lters and stored at À20 C. The cell-

45、free supernatants were tested for the presence of autoinducers that induce luminescence in V. harveyi reporter strain BB170, which has sensor 2but not sensor 1and is thus capable of sensing AI-2but not AI-1. In the bioassay, V. harveyi strain BB170was grown over-night at 30 C with aeration in autoin

46、ducer bioassay (ABbroth and then diluted 1:1000with AB medium (Bassler, Wright, Showalter, &Silverman, 1993. Next, 4.5mL of the diluted strain BB170and 500m L of the cell-free supernatant from each sample (V. parahaemolyticus supernatant from different temperatures grown on crab were added to 50

47、-mL Falcon tubes and shaken for 16h at 220rpm to allow luminescence formation by the reporter strain. Then, 100-m L samples were transferred to white microtiter plates and their luminescence was measured using a computer-controlled microplate luminometer (GloMax96Microplate Luminometer for Luminesce

48、nce; Promega, Madison, WI, USA. For the positive control, V. harveyi strain BB120(whichproduces AI-1and AI-2 was grown overnight at 30 C with shaking at 220rpm in LB broth, following which 1mL of cell-free supernatant from the culture was prepared as described above. 2.8. Field emission scanning ele

49、ctron microscopyField emission scanning electron microscopy (FESEMwas used to assess the bio lms formed on the SS, crab, and shrimp coupons, following previously described procedures (Jahid et al., 2013 with some modi cations. Each coupon was rinsed three times with PBS and the adhered cells were th

50、en xed in 4%glutaraldehyde (SigmaAldrich in PBS for 24h. The xed cells were serially treated with ethanol (50%for 15min, 60%for 15min, 70%for 15min, 80%for 15min, 90%for 15min, and then twice with 100%for 15min and then successively dehydrated with 33%,50%,66%,and 100%hex-amethyldisilazane (SigmaAld

51、rich in ethanol for 15min, respec-tively. The dehydrated samples were coated with platinum andobserved by FESEM. The electron microscope was operated at an accelerating voltage of 5kV with a 5-mm working distance.2.9. Statistical analysisAll treatments were conducted in three independent trials, wit

52、h each having triplicate samples. Data were analyzed by one-way analysis of variance using the Statistical Analysis System software (SASversion 9.2; SAS Institute Inc., Cary, NC, USA. Statistical sig-ni cance was considered at P <0.05.3. Results and discussion3.1. Quantitative bio lm formation as

53、say in microtiter plates Data on the ability of V. parahaemolyticus to produce bio lms on 96-well microtiter plates at 4, 10, 15, 20, 25, 30, and 37 C are shown in Table 1. Bio lms were formed at all the temperatures tested (theBFI increased with increasing temperature. The same increasing trend was

54、 found for bio lms formed on SS, crab, and shrimp sur-faces (Fig. 1. Bio lm formation was signi cantly stronger at 15e 37 C than at 4and 10 C. Depending on the environmental conditions (winterand summer range of storage temperature (5,10or 20 C may vary. During harvesting and processing of seafoods

55、for storage microbial pathogens and natural spoilage ora may grow, affected the composition and texture of seafood and poses a potential health risk to susceptible consumers (Mudoh, Parveen, Schwarz, Rippen, &Chaudhuri, 2014. The lowest temperatures at which V. parahaemolyticus has been reported

56、 to grow in vitro are 4 C (Wong, Chung, &Yu, 2002, 5 C (Twedt, 1989 and 8.3 C (Miles, Ross, Olley, &McMeekin, 1997, indicating that these or-ganisms can survive refrigeration temperatures and causes heat shock at 42 C on SS and glass surfaces (Wong et al., 2002. Ac-cording to the Internation

57、al Commission on Microbiological Spec-i cations for Foods (ICMSF,V. parahaemolyticus can grow rapidly in both broth and on seafood at temperatures ranging from 18to 40 C (ICMSF, 1996. Mizan et al. (2016reported that V. parahaemolyticus form strong bio lm on microtiter plate and SS surfaces at 30 C.

58、The bio lm formation might also be in uenced by the growth envi-ronment and stress conditions (Moltz &Martin, 2005; Nguyen, Yang, &Yuk, 2014. Rode, Langsrud, Holck, and Møretrø(2007indicated that bio lm formation of Staphylococcus aureus is stron-gest at suboptimal growth condition

59、s (46 C. Most strains of S. aureus have a growth optimum temperature of around 30 C in polystyrene microtiter plates.Table 1Bio lm formation index, exoprotease assay, and AI-2production of Vibrio para-haemolyticus at different temperatures (4e 37 C. Temperature ( C (BFI±SEM a (ng/mL±SEM (R

60、LU±SEM 40.21±0.015c 0.08±0.065c 6.89Â107±1.31Â104c 100.34±0.008c 0.14±0.16c 7.13Â107±2.15Â104c 150.52±0.023b 0.67±0.41b 8.71Â107±1.27Â104b 200.57±0.011b 0.62±0.54b 7.87Â107±1.63Â104b 250.64±0.041b 1.01±0.039a 7.94Â107±6.33Â104b 301.36±0.017a 1.16±0.47a 1.02Â108±2.16Â10