• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Effect of environmental stresses and high hydrostatic pressure on the Antibiotic susceptibility of pathogenic Vibrioparahaemolyticus

    2017-09-04 13:40:04OulimataNdiayeLIUHaiquanPANYingjieZHAOYong
    微生物學(xué)雜志 2017年3期
    關(guān)鍵詞:環(huán)境壓力溶血性弧菌

    Oulimata Ndiaye, LIU Hai-quan,2,3, PAN Ying-jie,2,3, ZHAO Yong,2,3*

    (1.College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306;2. Shanghai Engineering Research Centerof Aquatic-Product Processing & Preservation,Shanghai 201306;3. Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306)

    Effect of environmental stresses and high hydrostatic pressure on the Antibiotic susceptibility of pathogenicVibrioparahaemolyticus

    Oulimata Ndiaye1, LIU Hai-quan1,2,3, PAN Ying-jie1,2,3, ZHAO Yong1,2,3*

    (1.CollegeofFoodScienceandTechnology,ShanghaiOceanUniversity,Shanghai201306;2.ShanghaiEngineeringResearchCenterofAquatic-ProductProcessing&Preservation,Shanghai201306;3.LaboratoryofQuality&SafetyRiskAssessmentfor
    AquaticProductsonStorageandPreservation(Shanghai),MinistryofAgriculture,Shanghai201306)

    The aim of the present study was to determine the effect of environmental stresses (temperature, pH, osmotic pressure, high hydrostatic pressure (HHP)) on the antibiotic susceptibility of 12 different pathogenicVibrioparahaemolyticusisolates. Isolates were subjected to growth at (30 ℃and 37 ℃), an osmotic pressure of (1% and 6% NaCl), a media at pH (6.0 and 9.0) and three different HHP treatment (180, 250, 300 MPa). The minimal inhibitory concentrations (MICs) of tested antibiotics used against unstressed (control), stressed or post-stressed isolates were determined using the broth microdilution method. The study found that incubation under increased salt (6%), reduced salt (1%) and increased pH (9.0) conditions were commonly associated with increased antibiotic resistance. Incubation at 30 ℃ temperature, reduced pH (6.0) and HHP treatment were commonly associated with decreased antibiotic resistance. Besides, both ciprofloxacin and cefotaxime kept a constant MIC during almost all stress challenges. Thus our data demonstrate that exposure ofV.parahaemolyticusto some stress conditions may contribute on the rapid development of antibiotic resistance in this food borne pathogen.

    PathogenicVibrioparahaemolyticus; Antibiotic susceptibility; Environmental stresses; HHP

    Environmental stress can be defined as an external factor that has a negative impact on the physiological welfare of bacterial cells causing a reduction in growth rate and dramatically, an inhibition or death. These bactericidal stresses can be listed as extremes of temperature, osmotic pressure, pH, nutrient depletion[1], and also the use of toxic or inhibitory compounds like antibiotics[2]. During food processing bacteria get through a variety of conditions that may lead to chemical (acids, ethanol, alkaline, chlorine and salts) and physical (heat, radiation and pressure) stresses[3]. In this context, it is interesting to note that high hydrostatic pressure (HHP) can also inactivate microorganisms. In the food industry area, HHP is known to be the best alternative for thermal process in order to maintain the nutritional and functional values of food[4-7]. However, it has been shown that some cells can repair the sub-lethal damage induced by HHP, allowing them to proliferate once they have healed from the injury. Stress-inhibited bacteria act to retaliate the impact of environmental stresses by making phenotypic and genotypic adaptations[8]. Phenotypic alterations, including expression of protective shock proteins allow resistance to subsequent challenge with the same stress (stress hardening)[9], and thus are able to lead to cross-protection against a range of foreign challenges notably resistance to antibiotics[10-13]. It is currently believed that bacterial cells sense the actions of antibiotics just like any other form of environmental stress[2]. The mar (multiple antibiotic resistance) operon regulates the expression of a large variety of genes, including those coding for at least one broad-specificity efflux pump (the arcAB efflux pump), which are more strongly expressed under environmental stress conditions[14-16]. Stress alike oxidative stress, pH extremes, heat shock, osmotic shock, starvation, are known to be causing bacterial genomic reorganization or mutation[17], and such mechanisms of adaptive mutation have been related with the development of ABR[18]. This implies a direct linkage between environmental stresses happening in food industry and domestic environment[14], and the development of antibiotic resistance[19-20].Vibrioparahaemolyticusis part of the expanding group of water and food-borne pathogens. It is an important pathogen of shrimp, widely spread in the aquatic environment and increasingly causing significant economic problems within the aquaculture industry[21-22]. The factors ofV.parahaemolyticusmajor virulence are the thermostable direct hemolysin and the thermostable direct hemolysin-related hemolysin, encoded respectively the by thetdhandtrhgenes. The association between possession of thetdhgene by a strain and its faculty to cause gastroenteritis has been proved[23]. In the aquaculture field, the presence of pathogenicvibriosin shrimp ponds has brought farmers to look for effective control methods such as antibiotic therapy. Studies indicate that the antibiotic resistance ofvibriosisolated from penaeid culture environment is quite common[24-25], a fact that apparently constitutes a problem to this type of aquaculture activity[26]. In addition, there is the risk of environmental impact, since the use of antibacterial agents as prophylactic measure in aquaculture favors the selection of resistant bacteria, increasing the probability of transferring resistant genes to human pathogens and land animals[27]. A huge number of antibiotic resistant genes found in bacteria and environments can be listed as follows, β-lactam and penicillin resistant genespenA andblaTEM-1[28-29], chloramphenicol resistant genescatI,catII,catIII,catIV, andfloR[30], tetracycline resistant genestetA,tetB,tetC,tetD,tetE,tetG,tetH,tatJ,tatY,tatZ, and many more[31-33]. Via conjugation, transduction, or transformation, these antibiotic resistant gene can be transferred among different bacteria[34]. Antimicrobial resistant pathogenicV.parahaemolyticusis increasing exponentially and is expected to create a serious problem in the clinical treatment[35].Recently there have been some useful studies done to evaluate the change of antimicrobial susceptibility after exposure to certain stress and results nourish even more our concern and curiosity in that topic. Meng-Hsuan Lin et al[36]investigated the Susceptibility ofVibrioparahaemolyticusto disinfectants after prior exposure to sublethal stress. In our laboratory, mismatch between antimicrobial resistance phenotype and genotype of pathogenicV.parahaemolyticusisolated from seafood has been published[37]. Considering the importance of the increasing antibiotic-resistant bacteria in marine invertebrates intended for human consumption, this study aimed to determine the antibiotic susceptibility pattern of pathogenicV.parahaemolyticusstains isolated from sea food after exposure to different stress conditions.

    1 Materials and Methods

    1.1 Materials

    Bacterial strains As shown in Table 1, a total of 12 pathogenicV.parahaemolyticusisolates were used in this study. Two were American Type Culture Collection , ATCC 17802, ATCC 33847, and 10 were isolated from 4 types of seafood (Macrobrachiumnipponensis,PenaeusMonodon,PenaeusVannamei,Crassostreagigas), collected from wholesale markets during 2009 to 2013inShanghai.ThepresumptiveV.parahaemolyticusisolates were confirmed by the API 20E system (bioMérieux, Inc., Durham, NC, USA). Since previous reports have shown that the API 20E test was unreliable for the identification of theV.parahaemolyticusisolates[38-39], all strains were identified by the presence of the species-specifictlhgene using polymerase chain reaction (PCR) according to Kaysner and DePaola[40].

    Table 1 Sources and genotypes of pathogenic Vibrio parahaemolyticus isolates (n=12)

    1.2 Methods

    1.2.1 Preparation of antibiotic stock solutionsV.parahaemolyticusisolates were treated with 7 antibiotics (Table 2), commonly used in clinical therapy. These antimicrobials belongs to 3 classes which are: β-lactam (Amoxicillin-clavulanic acid: AMC, Cefotaxime: CTX), aminoglycoside (Amikacin: AK, Gentamincin: GN, Kanamycin: K, Streptomycin: S), quinolone (Ciprofloxacin: CIP). Antibiotic stock solutions were prepared following the manufacturer recommendations. All antibiotic stock solutions were sterilized using 0.20 mm disposable syringe filter units (ToyoRoshi Kaisha, Ltd., Tokyo, Japan) and serially diluted to provide the desired concentrations (0.01-1 000 μg/mL) in the reaction mixtures.

    Table 2 Minimal inhibition concentration (MIC) breakpoints used to determine antibiotic susceptibility[41]

    1.2.2 Preparation of unstressed (control) and stressed (temperature, pH, NaCl)V.parahaemolyticuscultures UnstressedV.parahaemolyticusisolates were cultured in Tryptic Soy Broth (Sigma Aldrich, France) supplemented with 3% NaCl at 37 ℃ for 18 h until they reached the stationary phase. The pH for that standard TSB solution was 7.5.V.parahaemolyticusstrains exposed to a low temperature stress were grown into a standard TSB solution. However the incubation temperature was 30 ℃ for 18 h. To examine the effect of post-low temperature condition on the susceptibility ofV.parahaemolyticustoward tested antibiotics, cells initially stressed by a growth at 30 ℃ were inoculated again into a standard TSB and incubated at 37 ℃ for 18 h. Two different levels of NaCl were used in TSB for the osmotic stress: 1% and 6% (wt/vol). The NaCl-supplemented suspensions were inoculated withV.parahaemolyticusand incubated at 37 ℃ for 18 h. Two different levels of pH were also used in the current study: 6.0. and 9.0. These pH Values were obtained by adding appropriate volumes of NaOH or HCL to TSB.V.parahaemolyticuscells grown from the different culture conditions were harvested by centrifugation (Herolab, UniCen M, Wiesloch, Germany) at 3 000 g for 10 min and pellets were resuspended and diluted to give a final concentration of approximately 5 log cfu/mL in the reaction mixtures for the antibiotic challenge.

    1.2.3 High hydrostatic pressure treatment Three different isolates were used for the HHP treatment:Macrobrachiumnipponensis(VPD8),Peanusmonodon(VPD33) andCrassostreagigas(VPD34). Strains were cultured in Tryptic Soy Broth (Sigma Aldrich, France) supplemented with 3% NaCl at 37 ℃ for 18 h until they reached stationary phase. Resulting cell suspensions were aseptically transferred to sterile polyethylene bags (internal length, 13 mm, and width, 6 mm; Yizuo Co., LTD., Taiwan) in 8 mL portions, vacuum packaged, and the bags were heat-sealed immediately. Assessment of the HHP treatment was conducted using a laboratory high-pressure processing system with a 2-L vessel (HHP. L2-600/2; Tianjin Sai Mei Int. Trade Co., Ltd, Tianjin, China). The bags containing samples were subjected to the pressure treatment in triplicate 180, 250 and 300 MPa, with a holding time of 5 min at room temperature around 25 ℃. After processing, the cultures treated with HHP were aseptically removed from the bags. Cultures were diluted to give a final concentration of approximately 5 log cfu/mL in the reaction mixtures for antibiotic challenge as described above. Cell suspensions were challenged by antibiotic exposure as described below.

    1.2.4 Antimicrobial assays (broth microdilution method) The minimal inhibitory concentration (MIC), defined as the lowest concentration of antibiotic required to inhibit visible growth of the test microorganisms, was determined using the broth micro-dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines[41]EscherichiacoliATCC 25922 was included as a quality control strain. To confirm the MIC, a 100 mL sample was taken at the end of incubation and viable cell numbers in visibly clear wells were assessed by plating on TSA. The MIC was confirmed if the viable cell numbers after incubation were not significantly different from the numbers at initial inoculation. The number of survivors was determined by plating on TSA before and after exposure to stress. Each antibiotic sensitivity trial was performed in triplicate. The breakpoint MICs of tested antibiotics againstV.parahaemolyticuswas interpretedaccordingto methods recommended by the CLSI[41].

    1.2.5 Statistical analysis Statistically significant differences (P<0.05) were established between control between control and test data were evaluated as recommended by Mann-Whitney U analysis using the Statistical Package for the Social Sciences (SPSS), version 11.0.

    2 Results

    2.1 The antibiotic resistance of unstressedVibrioparahaemolyticus

    All unstressed isolates (Table 3) used in this research were initially sensitive toward the antibiotics However, differential susceptibility was observed according to the type of antibiotic and also between isolates.

    Table 3 Changes in MIC of antibiotics applied to low temperature stressed V. parahaemolyticus strains

    Note (same as table four, five and six):“+” 0.5 to 2 fold increase in MIC (P<0.05);“-” 0.5 to 2 fold decrease in MIC (P<0.05);“++” 2.1 to 4-fold increase in MIC (P<0.05);“--” 2.1 to 4-fold increase in MIC (P<0.05);“+++”greater than 4-fold increase in MIC (P<0.05);“---” greater than 4-fold decrease in MIC (P<0.05);0 no change (P> 0.05);PS: post stress

    All strains showed the same susceptibility to cefotaxime and ciprofloxacine.Penaeusvannameiisolate(VPR512) and both,tdh,trhfrom commercial ATCC were more sensitive to gentamicin and amikacin. ATCC strains showed the higher sensitivity to amoxillin-clavicul acid (AMC) while themacrobrachiumnipponensisisolate (VPR101) and theCrassostreagigasisolate (VPD34) were less sensitive toward it. As a matter of fact this finding indicates different MIC susceptibility leaded with either the type of antibiotic or the type of isolate. The highest degree of inhibition was obtained when samples were treated with cefotaxime and ciprofloxacin. More than 90% of the isolates showed a growth inhibition after treatment with these two antibiotics in any of the applied stress conditions.

    2.2 The antibiotic resistance of temperature stressed and post-temperature stressedVibrioparahaemolyticus

    At 30 ℃, an increase in antibiotic susceptibility was noticed (Table 3). The MIC value was reduced especially for the streptomycin and kanamycin. However, the majority ofV.parahaemolyticusisolates had a MIC similar to those observed in the unstressed or control suspensions. Those antibiotics involve gentamicin, amikacin and particulary amoxillin-clavicul acid.Crassostreagigasisolate (VPD34) had lower MIC of amoxillin-clavicul acid. Further, this susceptibility decreased or remained constant after removal of the stress.

    2.3 The antibiotic resistance of pH stressedVibrioparahaemolyticus

    An increase in susceptibility was noticed at pH 6.0 (Table 4). Isolates exposed to amoxillin-clavicul acid and streptomycin was found to be less resistant at pH 6.0. Toward the rest of the antibiotics they showed a constant resistance and occasionally became less resistant. In contrast the resistance of the isolates raised up at pH 9.0 specially face to amikacin.Peanusmonodonisolate (VPD33) became resistant with a MIC raised up to 2 fold toward that antibiotic. Globally, the resistance increased or remained constant at pH 9.0.

    Table 4 Changes in MIC of antibiotics applied to acid-stressed V. parahaemolyticus strains

    4.2 預(yù)約優(yōu)選和排序功能 輸液時段設(shè)計是減少患者等候,均衡工作量的一個關(guān)鍵環(huán)節(jié)。我們廣泛聽取患者的意見和建議,總結(jié)手工預(yù)約的實踐經(jīng)驗,常規(guī)狀態(tài)下系統(tǒng)每天設(shè)置7個時間段,每50分鐘為1個時間段,2個時間段之間有10 min的緩沖期,每個時間段預(yù)約50例患者。根據(jù)患者喜好和輸液高峰時段的情況,系統(tǒng)默認(rèn)為患者選擇最早的時段,其中10:30~11:20時段只留給當(dāng)日就診后需要輸液的患者預(yù)約,也可以根據(jù)患者的需求自由選擇時間段。系統(tǒng)根據(jù)患者預(yù)約的先后為每個時段的患者自動排序。智能化的操作非常簡便、高效和人性化。這一功能的實現(xiàn) 也是“關(guān)愛患者,從細(xì)節(jié)做起”的體現(xiàn)。

    2.4 The antibiotic resistance of osmotic stressedVibrioparahaemolyticus

    Osmotic stressedV.parahaemolyticusisolates grown at 1% NaCl showed an increase of antibiotic resistance although with some isolate-antibiotic pairs, no significant change was detected (Table 5).

    2.5 The antibiotic resistance of post HHPVibrioparahaemolyticus

    An increase of antibiotic susceptibility was found according to the applied pressure treatment (Table 6). With a pressure of 180 MPa there was no significant difference between treated and untreatedV.parahaemolyticusisolates. However, a significant increase in susceptibility was noticed when the pressure was raised to 250 MPa.

    This was further increased when isolates wassubjected to 300 MPa at the same holding time which was 5 mn in all cases. For example, with a treatment at 180 MPaCrassostreagigasisolate (VPD34) had the same susceptibility as the control strain. Eventually its susceptibility increases by 2 fold at 250 MPa, and by 4 fold at 300 MPa. Nevertheless for some isolates-antibiotics couples, no change occurred after the HHP treatment. That was the situation of all isolates treated with gentamicine and theMacrobrachiumnipponensisisolate (VPD8) when treated with kanamycine.

    Table 5 Changes in MIC of antibiotics applied to osmotically stressed V. parahaemolyticus strains

    Note: A significant increase of resistance up to 2 fold was revealed when isolates such asPeanusmonodon(VPD33) andCrassostreagigas(VPD34) were treated by amikacin. Similar results were observed in isolates grown at 6% NaCl but the increase was expressed at comparatively lower levels in samples grown at 1% NaCl

    Table 6 Changes in MIC of antibiotics applied toV. parahaemolyticus strains survivors from HHP treatment

    3 Discussion

    Traditionally,vibriospecies are considered highly susceptible to the vast majority of antimicrobials[42]. However, antimicrobial resistance was reported in the recent decades and has evolved since, due to the unreasonable use of antimicrobials products in almost all systems reliable to living species[43-44]. An antibiotic might be expected to have variable effects on culture of genetically susceptible bacteria when the growth conditions allow different growth rates. Very few studies have addressed the impact of environment on the antibiotic susceptibility ofVibrioparahaemolyticus. To our knowledge, this is the first study noticing antibiotic resistance differences ofVibrioparahaemolyticusexposed to different kinds of stress. Nonetheless a number of studies have observed the differences in overallVibrioparahaemolyticusgrowth because of varying culture temperature[45-48]. For example, in bacteriological broth systems the growth ofV.parahaemolyticusin a temperature range between 8 to 45 ℃[49]and the growth rate and lag time from 10 to 30 ℃ were modeled[50]. In the current study, all strains were at first selected to be initially sensitive toward the antibiotics tested. When analyzing our results according to the antibiotic mechanism, we could notably observe that bacterial susceptibility was not influenced by the antibiotic class. Bacterial susceptibility toward ciprofloxacin and cefotaxime were very high compared to other antibiotics. However the mechanism of these bactericides is different, the cefotaxime act by the inhibition of bacterial cell wall synthesis while the Ciprofloxacin act by the inhibition of Protein Synthesis. The high sensitivity ofVibrioparahaemolyticustoward these two antibiotics was previously reported by Chan et al[51]. For the group of aminoglycoside antibiotic, they didn’t either give the same susceptibility pattern. Results were different from each antibiotic itself in all the stress conditions. Our study revealed an increase in antibiotic susceptibility ofV.parahaemolyticusin some cases when the organism was subjected to low temperature. A similar response has been reported inE.coli,S.entericaserovarTyphimurium, andS.Aureusgrown in conditions of continuing low-temperature stress by McMahon et al[52]. It has also been reported that increased antibiotic susceptibility was not maintained inE.colicultures when low-temperature stress was removed, nor inS.entericaserovarTyphimuriumorS.aureuscultures[52]. These temporary changes might be due to transient alterations affecting the bacterial cell wall or cell membranes. Modification of a normal environmental temperature can has adverse effect on the membrane fluidity. Consequently bacteria will make compensatory alterations in the degree of saturation of membrane fatty acids[53-56]to reestablish ideal states of membrane fluidity. However, more studies are needed to explore the process by which thermal stress can induce persistent increases in antibiotic susceptibility. In further support of the present observations, Salomon et al[8]found that T6SS1, highly regulated systems used by Gram-negative bacteria has an anti-bacterial activity under 23 ℃ and 30 ℃, but not at 37 ℃ in both high salt (MLB media) and low salt (LB media) conditions. Interestingly these findings closely match our results which showed in some cases a decrease of antibiotic resistance at 30 ℃. A high anti-Hcp response was also previously detected in cystic fibrosis patients with chronicP.aeruginosainfections[57], suggesting that Hcp is available for immune processing during bacterial infection. Furthermore, by reverting to our results, the minority of cases showing an increase of susceptibility at 30 ℃ might be explained if we relay to the fact that the expression of T6SS1 in free-living bacteria is minimal[58]. To our knowledge, that is the first evidence linking a component of a T6S system to antibiotic resistance. Even though the resistance mechanism is still a blur, it probably involves uncharacterized effectors of this secretion system.

    Ecological stresses such as osmotic, acid and cold shock provoke mutation in bacterial cells that has been reported to be related with the emergence of antibiotic resistance[18]. Under conditions of high salt, low salt or high pH stresses, the MICs of the antibiotic applied to most of our isolates were higher than the MICs of unstressed suspensions. McMahon et al[52]obtained the same results when the antibiotic resistance ofE.colisuspension was challenged in conditions of high-salt. However in contrast to our results in whichV.parahaemolyticuscells displayed a decrease of resistance, McMahon et al[52]reported an increase of resistance when the pH value was reduced. An increase greater than two fold was noticed in two isolates (VPD33 and VPD34) grown in medium with 1% NaCl and treated by amikacin. For example, isolates stressed at 1% NaCl showed in increase greater than that shown for isolates stressed at 6% NaCl. The resistance was also more pronounced with the couple, isolate (VPD33) and amikacin which showed once again an increase up to 2 fold at pH 9.0. In further support of the present observations, McMahon et al[52]found that an osmotic pressure more than 4.5% NaCl increased the antibiotic resistance ofE.coli,SalmonellaTyphimuriumandS.aureus. In addition, Ganjian et al[59]found that NaCl concentrations up to 35% augmented the antibiotic resistance ofS.aureuscells.

    Reaction face to acid stress is an intricate phenomenon. Acid stress can be described as the combined biological effect of low pH and weak (organic) acids, such as acetate, propionate and lactate present in the environment (food) as a result of fermentation, or alternatively, when added as preservatives[60-62]. Exposure to acid stress may enhance bacterial pathogenesis by altering the virulence-associated characteristics of acid adapted cells. The reduced cytotoxicity of acid-adapted cell could be a consequence of physical injury.V.choleraecells grown at pH 6.0 had reduced expression of various enzymes necessary for lipopolysaccharide biosynthesis, leading to deterioration in the external membrane and an increased susceptibility to hydrophobic drugs like erythromycin[63].

    In our study, an increase of susceptibility was noticed following the pressure treatments (180 MPa, 250 MPa) and became more dramatic when the applied pressure was 300 MPa. Damages from HHP treatment with pressure level setting similar to ours is well documented in the literature. HHP causes inhibition of protein synthesis and reversible protein denaturation at 50 MPa-100 MPa, membrane damage at 200 MPa, and irreversible protein denaturation at ≥300 MPa[64-66]. These results demonstrated increasing changes in cell morphology with increasing pressure treatment. To our knowledge, the present study is the first to assess the effects of post HHP treatment on the antibiotic susceptibility of microorganisms. The inactivating effects of HHP may be facilitated by the presence of antimicrobial agents[45]. Because Bacteriocins do require a given incubation time to cause cell damage their bactericidal effect in combination with an HHP treatment are more pronounced Some studies have been reported in the combination of antimicrobial and HHP treatments of lower intensity (500 MPa, 5 min). The inactivation of staphylococci in pudding by nisin improved when combined with HHP. Viable counts reductions significantly increased by 0.87 log cycle cycle for the 500 UI/g nisin-HHP combination comparing to the single HHP treatment. Nisin was again tested singly or in combination with HHP treatment (500 MPa, 5 min) for staphylococci in pudding during one week storage at 4 ℃. The single addition of nisin dramatically (P<0.005) reduces viable counts of staphylococci, in rice pudding[67].

    Another research has found that the addition of antibiotics to the medium that should be used for HHP treatment can improve the disinfection efficacy of HHP treatment. Firstly, a variety of bacteria were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes gave satisfying but incomplete elimination especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of the inactivation[45].

    This part of the study was to evaluate ifV.parahaemolyticuscells that have survived HHP treatment could develop antibiotic resistance or become more susceptible.V.parahaemolyticusselected in this study, did not develop any antibiotic resistance after the stress from HHP treatments. The MICs of the antibiotics applied to each isolates were lower than the MICs for (control) unstressed organisms depending on the applied pressure. Our findings suggest also that combination of HHP and antibiotics could improve the efficacy of HHP treatment. Despite the fact that only three samples were used for each of the combinations of antibiotics and HHP, conclusions regarding the superiority of one combination to one over could also be drawn from this study.

    This study made it possible to investigate various effects of stress on the antibiotic susceptibility ofV.parahaemolyticuscells. It was found that incubation under some type of stress (30 ℃ and reduced pH) may increase bacterial susceptibility while exposure to some others (increased pH, increased and reduced salt) may have the adverse effect. In general, the decrease in antibiotic resistance at low temperature stress was not maintained after removal of the stress. Our research showed that no increase of resistance was found in the antibiotic susceptibility testing after HHP treatments. This suggests that exposure ofV.parahaemolyticusto certain stress conditions may contribute on the rapid development of antibiotic resistance in this food borne pathogen. However more studies are warranted to validate these results and further understand the mechanisms underlying variations in antibiotic susceptibility.

    Reference

    國家自然科學(xué)基金面上項目(31271870, 31571917);上海水產(chǎn)品加工及貯藏工程技術(shù)研究中心(11DZ2280300);

    Oulimata Ndiaye 女,碩士研究生。研究方向為食品安全。 E-mail: olitaah@live.com

    環(huán)境壓力條件與超高壓對致病性副溶血性弧菌耐藥性的影響

    Oulimata Ndiaye1, 劉海泉1,2,3, 潘迎捷1,2,3, 趙 勇1,2,3*

    (1.上海海洋大學(xué) 食品學(xué)院,上海 201306;2.上海水產(chǎn)品加工及貯藏工程技術(shù)研究中心,上海 201306;3.農(nóng)業(yè)部貯藏保鮮質(zhì)量安全風(fēng)險評估實驗室(上海),上海 201306)

    分析不同環(huán)境壓力條件(溫度、pH、滲透壓和超高壓)對12株致病性副溶血性弧菌耐藥性的影響。利用微量肉湯稀釋法測定菌株在不同溫度(37 ℃和30 ℃)、滲透壓(1%和 6% NaCl)、pH(6.0和9.0)及超高壓(180、250和300 MPa)條件影響下,其對所測抗生素的最小抑菌濃度(MIC)。結(jié)果表明,在1%和6% NaCl質(zhì)量分?jǐn)?shù)、pH 9.0條件下,副溶血性弧菌的耐藥性增強(qiáng),而在30 ℃、pH 6和超高壓條件影響下,其耐藥性減弱。此外,菌株在所測環(huán)境壓力條件影響下,其對環(huán)丙沙星和頭孢類抗生素的耐藥性基本保持不變。環(huán)境壓力條件的改變可能會增強(qiáng)致病性副溶血性弧菌的耐藥性。

    致病性副溶血性弧菌;耐藥性;環(huán)境壓力;超高壓

    Q93-331;Q939.11+8

    A

    1005-7021(2017)03-0065-12

    * 通訊作者。男,博士,教授。研究方向為食品安全學(xué)。Tel: 021-61900503, E-mail: yzhao@shou.edu.cn

    10.3969/j.issn.1005-7021.2017.03.012

    Funds Project: The National Natural Science Foundation of China (31271870, 31571917); Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation (11DZ2280300);Supported by Innovation Program of Shanghai Municipal Education Commission

    Author profile: Oulimata Ndiaye, female, master, majoring in food safety. E-mail: olitaah@live.com

    *Corresponding author:male, professor. majoring in food safety. Tel: 021-61900503, E-mail: yzhao@shou.edu.cn

    Draft accepted date:2016-05-18;Revision returned date:2016-07-30

    上海市教育委員會科研創(chuàng)新計劃項目

    猜你喜歡
    環(huán)境壓力溶血性弧菌
    銷量增長200倍!“弧菌克星”風(fēng)靡行業(yè),3天殺滅98%弧菌
    副溶血弧菌檢測方法的研究進(jìn)展
    碳氧血紅蛋白在新生兒ABO溶血性黃疸中的臨床意義
    故障狀態(tài)下純電動汽車環(huán)境壓力及海拔高度估算方法
    北京汽車(2021年1期)2021-03-04 13:05:46
    可替換牙刷
    如何有效防控對蝦養(yǎng)殖中的弧菌病
    副溶血弧菌噬菌體微膠囊的制備及在餌料中的應(yīng)用
    利巴韋林片致溶血性貧血伴急性腎衰竭1例
    去白細(xì)胞懸浮紅細(xì)胞儲血袋兩種放置方式的溶血性分析
    貴陽市經(jīng)濟(jì)發(fā)展與環(huán)境壓力實證分析
    91精品伊人久久大香线蕉| 美女视频免费永久观看网站| 久热这里只有精品99| 亚洲经典国产精华液单| 成年女人在线观看亚洲视频| 免费av不卡在线播放| 国产一区二区三区av在线| 国国产精品蜜臀av免费| 在线 av 中文字幕| 极品少妇高潮喷水抽搐| 夫妻性生交免费视频一级片| 国产精品成人在线| 国产视频首页在线观看| 最近2019中文字幕mv第一页| 国产欧美日韩精品一区二区| 交换朋友夫妻互换小说| 国产亚洲欧美精品永久| 毛片女人毛片| 亚洲国产毛片av蜜桃av| 午夜福利在线观看免费完整高清在| 久久精品国产自在天天线| 久久ye,这里只有精品| 久久久久网色| tube8黄色片| 99视频精品全部免费 在线| 日韩不卡一区二区三区视频在线| 久久 成人 亚洲| 婷婷色av中文字幕| 一个人看视频在线观看www免费| 亚洲精品日韩在线中文字幕| 亚洲精品国产色婷婷电影| 下体分泌物呈黄色| 国产色婷婷99| 国产女主播在线喷水免费视频网站| 18禁裸乳无遮挡动漫免费视频| 欧美xxxx黑人xx丫x性爽| 亚洲精品国产av蜜桃| 一个人看视频在线观看www免费| 国产精品成人在线| 视频中文字幕在线观看| 亚洲精品456在线播放app| 中国美白少妇内射xxxbb| 亚洲精品乱码久久久久久按摩| 一级片'在线观看视频| 春色校园在线视频观看| 免费少妇av软件| 永久网站在线| 国产国拍精品亚洲av在线观看| 国产在视频线精品| 国产成人a区在线观看| 另类亚洲欧美激情| 亚洲av日韩在线播放| 日本色播在线视频| 欧美精品国产亚洲| 国产 精品1| 在线观看免费视频网站a站| 欧美xxⅹ黑人| 国产男人的电影天堂91| 国产精品久久久久久精品电影小说 | 亚洲人与动物交配视频| 国产淫语在线视频| 亚洲第一区二区三区不卡| 丰满人妻一区二区三区视频av| 日韩欧美精品免费久久| 亚洲人成网站高清观看| 亚洲精品aⅴ在线观看| 欧美精品国产亚洲| 日韩 亚洲 欧美在线| 国产欧美日韩精品一区二区| 国产极品天堂在线| 久久综合国产亚洲精品| 欧美成人午夜免费资源| 欧美三级亚洲精品| 九色成人免费人妻av| 在线免费十八禁| 一级毛片aaaaaa免费看小| 国产免费一级a男人的天堂| 又粗又硬又长又爽又黄的视频| 日韩亚洲欧美综合| 另类亚洲欧美激情| 久久精品国产自在天天线| 男男h啪啪无遮挡| 久久精品国产a三级三级三级| 一个人看的www免费观看视频| 一区二区av电影网| 最近手机中文字幕大全| 免费少妇av软件| 国产高潮美女av| 精品久久久久久久久av| 日韩一区二区三区影片| 看免费成人av毛片| 国产成人91sexporn| 午夜老司机福利剧场| 老师上课跳d突然被开到最大视频| 99视频精品全部免费 在线| 国产欧美另类精品又又久久亚洲欧美| 一本一本综合久久| 亚洲精华国产精华液的使用体验| 亚洲精品日韩av片在线观看| 丰满少妇做爰视频| 成人美女网站在线观看视频| 中文字幕av成人在线电影| 亚洲精品国产成人久久av| 国产精品嫩草影院av在线观看| 午夜精品国产一区二区电影| 韩国高清视频一区二区三区| 99热6这里只有精品| 乱码一卡2卡4卡精品| 搡老乐熟女国产| 国产色婷婷99| 久久婷婷青草| 大香蕉久久网| 国产黄频视频在线观看| 久久久久视频综合| 日韩欧美精品免费久久| 久热久热在线精品观看| 在线观看人妻少妇| 五月开心婷婷网| av在线观看视频网站免费| 免费av中文字幕在线| 97热精品久久久久久| 国产黄色视频一区二区在线观看| 国产精品久久久久久精品古装| 亚洲第一av免费看| 永久网站在线| 日日啪夜夜撸| 国产欧美另类精品又又久久亚洲欧美| 美女福利国产在线 | 极品教师在线视频| 久久久欧美国产精品| 国内少妇人妻偷人精品xxx网站| 中文精品一卡2卡3卡4更新| 亚洲四区av| 一个人免费看片子| 精品国产露脸久久av麻豆| 在线观看一区二区三区| 久久精品久久精品一区二区三区| 国产视频内射| 亚洲丝袜综合中文字幕| 午夜福利在线观看免费完整高清在| 亚洲伊人久久精品综合| 国产av国产精品国产| 欧美3d第一页| 九草在线视频观看| 一区在线观看完整版| 久久影院123| 最近手机中文字幕大全| 中文在线观看免费www的网站| 国产精品一区二区三区四区免费观看| 成人黄色视频免费在线看| 亚洲熟女精品中文字幕| 视频中文字幕在线观看| 亚洲欧美日韩另类电影网站 | 国产精品人妻久久久久久| 中文字幕av成人在线电影| 欧美日韩视频精品一区| 夫妻午夜视频| 99热6这里只有精品| 久久精品熟女亚洲av麻豆精品| 久久久久久久久久人人人人人人| 精品久久久久久久久亚洲| 丰满乱子伦码专区| 狂野欧美白嫩少妇大欣赏| 国产免费又黄又爽又色| 日本免费在线观看一区| 亚洲av.av天堂| 成人无遮挡网站| 亚洲图色成人| 国产白丝娇喘喷水9色精品| 亚洲欧美成人综合另类久久久| 综合色丁香网| 欧美成人精品欧美一级黄| 亚洲欧美成人精品一区二区| 久久99精品国语久久久| 伦理电影免费视频| 国产成人aa在线观看| 亚洲第一av免费看| 在线天堂最新版资源| 国产成人免费观看mmmm| 亚洲国产最新在线播放| 久久精品国产鲁丝片午夜精品| 久久鲁丝午夜福利片| 亚洲精品日韩av片在线观看| 成人国产av品久久久| 亚洲欧美成人精品一区二区| 欧美+日韩+精品| 国产成人精品久久久久久| 一级毛片 在线播放| 久久久久久久精品精品| 美女视频免费永久观看网站| 日韩国内少妇激情av| 免费人妻精品一区二区三区视频| 久久久久人妻精品一区果冻| 欧美成人一区二区免费高清观看| 性色av一级| 大又大粗又爽又黄少妇毛片口| 成人亚洲欧美一区二区av| 亚洲怡红院男人天堂| 六月丁香七月| 三级国产精品片| 欧美一级a爱片免费观看看| 亚洲色图av天堂| 日韩中字成人| 中国国产av一级| 狂野欧美激情性bbbbbb| 日韩电影二区| 成年美女黄网站色视频大全免费 | 日韩欧美一区视频在线观看 | 精品人妻视频免费看| 视频中文字幕在线观看| 亚洲国产精品成人久久小说| 免费看日本二区| 精品亚洲成a人片在线观看 | 色哟哟·www| 亚洲成人手机| 亚洲精品成人av观看孕妇| 国产成人精品一,二区| 老司机影院毛片| 精品国产乱码久久久久久小说| 成人影院久久| 乱系列少妇在线播放| 最近最新中文字幕大全电影3| 看十八女毛片水多多多| 小蜜桃在线观看免费完整版高清| 久久热精品热| 伦理电影免费视频| 在线免费观看不下载黄p国产| 国产日韩欧美在线精品| 欧美日韩综合久久久久久| 精品久久久久久电影网| 久久青草综合色| 嘟嘟电影网在线观看| 亚洲国产精品999| 日韩欧美 国产精品| 日韩av免费高清视频| 国产精品一区www在线观看| 国产色婷婷99| 蜜臀久久99精品久久宅男| 舔av片在线| 老女人水多毛片| 国产黄频视频在线观看| 亚洲欧美一区二区三区国产| 国产精品一区二区在线观看99| 国产精品三级大全| 97在线人人人人妻| 精品久久久久久久久亚洲| 亚洲欧美一区二区三区国产| 成人亚洲欧美一区二区av| 久久综合国产亚洲精品| 免费看不卡的av| 一本—道久久a久久精品蜜桃钙片| 男人狂女人下面高潮的视频| 男人舔奶头视频| 能在线免费看毛片的网站| 熟女av电影| 亚州av有码| 国产91av在线免费观看| av在线观看视频网站免费| 久久久色成人| 免费观看在线日韩| 性色avwww在线观看| 国产精品99久久久久久久久| 精品一区在线观看国产| 中文字幕久久专区| 我的女老师完整版在线观看| 久久精品国产亚洲av天美| av播播在线观看一区| 中文乱码字字幕精品一区二区三区| 久久久久久久久久久免费av| 国产高清有码在线观看视频| 亚洲国产精品999| 大片电影免费在线观看免费| 国产大屁股一区二区在线视频| 欧美日韩在线观看h| 在线免费十八禁| 人体艺术视频欧美日本| 少妇裸体淫交视频免费看高清| 亚洲第一av免费看| 97超视频在线观看视频| 欧美高清性xxxxhd video| 久久久午夜欧美精品| 在现免费观看毛片| av天堂中文字幕网| 亚洲成人手机| 看十八女毛片水多多多| 久久6这里有精品| 日韩欧美一区视频在线观看 | 国产精品久久久久成人av| 五月开心婷婷网| 18禁在线播放成人免费| 国产精品不卡视频一区二区| 欧美少妇被猛烈插入视频| 国产亚洲av片在线观看秒播厂| 久久国产乱子免费精品| 天堂俺去俺来也www色官网| av网站免费在线观看视频| 能在线免费看毛片的网站| 黄色配什么色好看| av国产精品久久久久影院| 欧美日本视频| 狂野欧美激情性xxxx在线观看| 亚洲三级黄色毛片| av又黄又爽大尺度在线免费看| 直男gayav资源| 观看av在线不卡| 在线免费观看不下载黄p国产| 国产精品一区二区性色av| 午夜老司机福利剧场| 伦理电影大哥的女人| 欧美日韩视频高清一区二区三区二| a级一级毛片免费在线观看| 亚洲国产欧美在线一区| 国产高清三级在线| 97超视频在线观看视频| 99热这里只有是精品50| 国产精品国产三级国产av玫瑰| 成年av动漫网址| 亚洲欧美日韩无卡精品| 蜜桃亚洲精品一区二区三区| 日本午夜av视频| 免费在线观看成人毛片| 日产精品乱码卡一卡2卡三| 国产精品久久久久久精品电影小说 | 久久久久视频综合| 高清在线视频一区二区三区| 99热网站在线观看| 精品亚洲乱码少妇综合久久| 精品一区二区免费观看| 久久久久久久国产电影| 亚洲av成人精品一二三区| 国产精品久久久久久精品古装| 久久久久久九九精品二区国产| 欧美zozozo另类| 亚洲av在线观看美女高潮| 插逼视频在线观看| 少妇人妻久久综合中文| 久久久久网色| 亚洲人成网站在线观看播放| 少妇丰满av| a 毛片基地| www.av在线官网国产| 久久久久久久久大av| 秋霞在线观看毛片| 黄色一级大片看看| 日本猛色少妇xxxxx猛交久久| 蜜桃亚洲精品一区二区三区| 插逼视频在线观看| 中国国产av一级| 国产 一区 欧美 日韩| 国产精品偷伦视频观看了| 日韩免费高清中文字幕av| 另类亚洲欧美激情| 亚洲av成人精品一二三区| 熟女人妻精品中文字幕| h日本视频在线播放| 国产精品精品国产色婷婷| 亚洲av中文字字幕乱码综合| 亚洲国产欧美人成| 亚洲精华国产精华液的使用体验| 偷拍熟女少妇极品色| 久久av网站| 亚洲国产精品一区三区| 中国美白少妇内射xxxbb| 久久影院123| 久久亚洲国产成人精品v| 中国国产av一级| 日日摸夜夜添夜夜添av毛片| 久久久久久久精品精品| 女性生殖器流出的白浆| 99久久综合免费| 大香蕉久久网| 能在线免费看毛片的网站| 最近中文字幕2019免费版| 王馨瑶露胸无遮挡在线观看| 日本免费在线观看一区| av女优亚洲男人天堂| 边亲边吃奶的免费视频| 国产老妇伦熟女老妇高清| 国产女主播在线喷水免费视频网站| 精品一区二区三区视频在线| 男人舔奶头视频| 久久久久性生活片| 久久国产乱子免费精品| 欧美成人午夜免费资源| 中国三级夫妇交换| 国产免费又黄又爽又色| 丰满乱子伦码专区| 99国产精品免费福利视频| 在线观看美女被高潮喷水网站| 精品少妇黑人巨大在线播放| 国产成人a∨麻豆精品| 色综合色国产| 成人漫画全彩无遮挡| 一级毛片aaaaaa免费看小| 国产亚洲午夜精品一区二区久久| 少妇的逼水好多| 国产高清国产精品国产三级 | 久久 成人 亚洲| h日本视频在线播放| 国模一区二区三区四区视频| 男女免费视频国产| 久久久久国产精品人妻一区二区| 国产午夜精品久久久久久一区二区三区| 韩国高清视频一区二区三区| 久久精品国产自在天天线| 国产久久久一区二区三区| 国产伦精品一区二区三区视频9| 丝瓜视频免费看黄片| 亚洲av男天堂| 丝袜喷水一区| 日韩av免费高清视频| 小蜜桃在线观看免费完整版高清| 观看美女的网站| 国产一区二区三区综合在线观看 | 大话2 男鬼变身卡| 在线免费观看不下载黄p国产| 国产亚洲5aaaaa淫片| 男人狂女人下面高潮的视频| 久久99热这里只有精品18| 国产精品麻豆人妻色哟哟久久| 中文精品一卡2卡3卡4更新| 久热这里只有精品99| 亚洲精品国产成人久久av| 亚洲,欧美,日韩| 日本爱情动作片www.在线观看| 久久久久久久久久久免费av| av不卡在线播放| 22中文网久久字幕| 男人狂女人下面高潮的视频| 亚洲伊人久久精品综合| 精品一区二区三卡| 狂野欧美激情性xxxx在线观看| 久久久久久久久久成人| 91久久精品国产一区二区成人| 国产av精品麻豆| 99久久精品国产国产毛片| 男的添女的下面高潮视频| 久久久久国产精品人妻一区二区| av免费观看日本| 国产乱人偷精品视频| 亚洲av福利一区| 人人妻人人看人人澡| 天美传媒精品一区二区| h日本视频在线播放| 99久久精品国产国产毛片| 国产永久视频网站| 国产一级毛片在线| 亚洲伊人久久精品综合| h视频一区二区三区| 国产精品爽爽va在线观看网站| 十八禁网站网址无遮挡 | 国产日韩欧美亚洲二区| 精品久久国产蜜桃| 免费看光身美女| 天天躁日日操中文字幕| 国产女主播在线喷水免费视频网站| 99热6这里只有精品| 亚洲成人中文字幕在线播放| 国产 一区 欧美 日韩| 亚洲欧洲日产国产| 亚洲国产日韩一区二区| 香蕉精品网在线| 日本色播在线视频| 老女人水多毛片| 97在线视频观看| 国产伦在线观看视频一区| 国产av国产精品国产| 高清午夜精品一区二区三区| 欧美精品亚洲一区二区| 观看av在线不卡| 老师上课跳d突然被开到最大视频| 有码 亚洲区| 亚洲在久久综合| 一级毛片我不卡| 日韩欧美 国产精品| 妹子高潮喷水视频| 国产成人91sexporn| 精品一品国产午夜福利视频| 久久综合国产亚洲精品| 最近中文字幕2019免费版| 人妻少妇偷人精品九色| 最黄视频免费看| 精品亚洲成a人片在线观看 | 国产黄片美女视频| 亚洲国产色片| 一区二区三区精品91| 国产欧美日韩一区二区三区在线 | 3wmmmm亚洲av在线观看| 免费少妇av软件| 成人漫画全彩无遮挡| 久久女婷五月综合色啪小说| 国产成人aa在线观看| 精品久久久久久久久亚洲| 久久久久久久亚洲中文字幕| 我要看日韩黄色一级片| 黑丝袜美女国产一区| 精品一区二区三区视频在线| 日本午夜av视频| 久久久久久久亚洲中文字幕| 久久人人爽av亚洲精品天堂 | 精品人妻熟女av久视频| 日韩中字成人| 联通29元200g的流量卡| 日本爱情动作片www.在线观看| 一级毛片黄色毛片免费观看视频| 在线免费观看不下载黄p国产| 视频区图区小说| 成年女人在线观看亚洲视频| 国精品久久久久久国模美| 国产免费福利视频在线观看| 干丝袜人妻中文字幕| 十分钟在线观看高清视频www | 五月开心婷婷网| 久久久久网色| 美女主播在线视频| 欧美97在线视频| 精品少妇久久久久久888优播| 久久久久久久久久久免费av| 大片免费播放器 马上看| 老司机影院毛片| 亚洲va在线va天堂va国产| 网址你懂的国产日韩在线| 啦啦啦视频在线资源免费观看| 人人妻人人爽人人添夜夜欢视频 | 亚洲av男天堂| 中文在线观看免费www的网站| 日日撸夜夜添| 国产精品麻豆人妻色哟哟久久| 国产一区亚洲一区在线观看| 成人综合一区亚洲| 国产精品免费大片| 欧美变态另类bdsm刘玥| 激情 狠狠 欧美| 99re6热这里在线精品视频| 最近中文字幕高清免费大全6| 国产欧美亚洲国产| 日日摸夜夜添夜夜爱| 亚洲av二区三区四区| 日韩欧美精品免费久久| 欧美精品一区二区大全| 精品99又大又爽又粗少妇毛片| 自拍欧美九色日韩亚洲蝌蚪91 | 精品国产乱码久久久久久小说| 日韩电影二区| 男男h啪啪无遮挡| 亚洲精品视频女| av黄色大香蕉| 超碰av人人做人人爽久久| 全区人妻精品视频| 啦啦啦啦在线视频资源| 爱豆传媒免费全集在线观看| 国产在线视频一区二区| 简卡轻食公司| 99久久精品热视频| 赤兔流量卡办理| 国产精品久久久久久av不卡| 这个男人来自地球电影免费观看 | 亚洲人成网站在线播| 亚洲内射少妇av| 成人亚洲精品一区在线观看 | 亚洲欧美精品自产自拍| 久热久热在线精品观看| 日本猛色少妇xxxxx猛交久久| h日本视频在线播放| 久久精品国产亚洲网站| 亚洲精品乱久久久久久| 亚洲最大成人中文| 国产精品久久久久久精品电影小说 | 最近的中文字幕免费完整| 少妇人妻 视频| 亚洲欧美成人精品一区二区| 我要看黄色一级片免费的| av免费观看日本| 秋霞伦理黄片| 日韩欧美精品免费久久| 久久国产乱子免费精品| 国产欧美日韩精品一区二区| 欧美成人一区二区免费高清观看| 欧美精品一区二区免费开放| 在线观看一区二区三区激情| 亚洲aⅴ乱码一区二区在线播放| 国内少妇人妻偷人精品xxx网站| 伊人久久国产一区二区| 青春草亚洲视频在线观看| 欧美xxⅹ黑人| 欧美高清性xxxxhd video| 青春草亚洲视频在线观看| 寂寞人妻少妇视频99o| 免费黄网站久久成人精品| 女性被躁到高潮视频| 最新中文字幕久久久久| 日韩在线高清观看一区二区三区| 一级毛片我不卡| 一区在线观看完整版| 亚洲av中文字字幕乱码综合| 性色avwww在线观看| 亚洲成人中文字幕在线播放| 赤兔流量卡办理| 国产精品久久久久成人av| 日韩伦理黄色片| 久久亚洲国产成人精品v| 女性被躁到高潮视频| 亚洲欧洲国产日韩| 蜜桃在线观看..| 男人和女人高潮做爰伦理| 视频区图区小说| 久久av网站| 简卡轻食公司| 我的女老师完整版在线观看| 久久久久国产网址| 少妇 在线观看| 97超视频在线观看视频| 女人久久www免费人成看片| 久久99热6这里只有精品| 九色成人免费人妻av|