Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H₂O₂ compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Contingency locus in ctsR of Listeria monocytogenes Scott A: a strategy for occurrence of abundant piezotolerant isolates within clonal populations

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10(-5). Subsequent sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n = 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within L. monocytogenes cultures, thus playing an important role in survival.     

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance,stress resistance,motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.     

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.     

Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices

The objective of this study was to compare high pressure resistance of Listeria monocytogenes strains at 25 degrees C and 50 degrees C at 350 MPa and to use high pressure (250 MPa and 350 MPa) at 30 degrees C and 40 degrees C for the inactivation of the relatively most pressure resistant strain inoculated in pasteurized apple, apricot, cherry and orange juices. L. monocytogenes CA was found to be the relatively most pressure resistant strain and increasing pressurization from 250 MPa to 350 MPa at 30 degrees C had an additional three to four log cycle reduction in viability, still leaving viable cells after 5 min. When 350 MPa at 40 degrees C for 5 min was applied more than eight log cycle reduction in cell population of all fruit juices was achieved. This study demonstrated that low temperature (40 degrees C) high pressure (350 MPa) treatment has the potential to inactivate relatively pressure resistant L. monocytogenes strains inoculated in different fruit juices within 5 min.     

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage

Bacteria are expected to be injured or killed by high hydrostatic pressure (HHP). This depends on pressure levels, species and strain of the microorganism and subsequent storage. Injured bacteria may be repaired which could affect the microbiological quality of foodstuffs with an important safety consideration especially in low acid food products. In this study two Gram-positive (Listeria monocytogenes CA and Staphylococcus aureus 485) and two Gram-negative (Escherichia coli O157:H7 933 and Salmonella enteritidis FDA) relatively pressure resistant strains of foodborne pathogens were pressurized at 350, 450 and 550 MPa in milk (pH 6.65) and stored at 4, 22 and 30 degrees C. The results of shelf life studies indicated two types of injury, I1 and I2, for all the pathogens studied. It is obvious that I2 type injury is a major injury and after its repair (I2 to I1), the cells can form colonies on non-selective but not on selective agar. The formation of colonies on both selective and non-selective agar occurs only after full recovery of injury (I1 to AC). The results presented in this study show that even if injured cells are not detected immediately after HHP treatment, I2 type injury could be potentially present in the food system. Therefore, it is imperative that shelf life studies must be conducted over a period of time for potential repair of I2 type injury either to detectable injury (I1) or to active cells (AC) to ascertain microbiological safety of low acid food products.     

Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy

Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min. Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L. monocytogenes, than in the other test micro-organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.     

Isolation of highly heat-resistant Listeria monocytogenes variants by use of a kinetic modeling-based sampling scheme

Stable high-hydrostatic-pressure (HHP)-resistant Listeria monocytogenes LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their ctsR genes. For the variants whose ctsR genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant L. monocytogenes variants.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Regulation of Cytochrome c- and Quinol Oxidases,and Piezotolerance of Their Activities in the Deep-Sea Piezophile Shewanella violacea DSS12 in Response to Growth Conditions

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O₂ concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O₂ concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O₂ concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.     

Piezotolerant Small-Colony Variants with Increased Thermotolerance, Antibiotic Susceptibility, and Low Invasiveness in a Clonal Staphylococcus aureus Population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.     

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions

Aim:  The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated.
Methods and Results:  Two clinical strains harvested in stationary phase were pressurized at 20°C and 37°C within a range of 50–400 MPa, in a phosphate (pH 7·0) or a citrate phosphate buffer (pH 5·6), for 10 min. Treatment efficiencies were determined by logarithmic comparisons of culturable cells on blood agar before and after treatment. Results were statistically compared using an anova of culturable cells after treatment to evaluate the effect of all factors. At least a 7-log reduction in cell numbers was observed for both strains. The pH and the strains had no effect on HHP treatment at 20°C while at 37°C, both pH and strain influenced significantly the HHP treatment on C. jejuni .
Conclusions:  The pressure efficacy on C. jejuni eradication was affected by both environmental and biological factors.
Significance and Impact of the Study:  Depending on the treatment conditions, C. jejuni sensitivity to HHP can significantly vary. The determination of the inactivation treatment by HPP has to be normalized considering the interaction of environmental and biological factors.     

中性体系中超高压－Nisin协同作用下的细菌致死机理

摘要：【目的】为保证超高压中性食品的杀菌强度,可以??????????通过添加Nisin等细菌素协同杀菌以达到商业无菌要求。本文从分子水平和超微结构揭示二者协同作用下的细胞致死机理,为超高压杀菌在中性食品中的应用奠定理论基础。【方法】采用pH7.0的环境体系,100－500 MPa的超高压处理,Nisin浓度为200 IU/mL。通过荧光染色法和紫外吸收法检测细胞膜通透性,傅里叶转换红外光谱法检测细菌细胞壁、蛋白以及核酸的变化,透射电镜观察细菌在协同作用下的形态变化。【结果】结果发现：中性条件下,超高压与     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices

AIMS: The aim of the study was to investigate the combined antimicrobial action of the plant-derived volatile carvacrol and high hydrostatic pressure (HHP). METHODS AND RESULTS: Combined treatments of carvacrol and HHP have been studied at different temperatures, using exponentially growing cells of Listeria monocytogenes, and showed a synergistic action. The antimicrobial effects were higher at 1 degrees C than at 8 or 20 degrees C. Furthermore, addition of carvacrol to cells exposed to sublethal HHP treatment caused similar reductions in viable numbers as simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with L. monocytogenes. CONCLUSION: Carvacrol and HHP act synergistically and the antimicrobial effects of the combined treatment are greater at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP and indicates the potential of these combined treatments in food processing.     

Salinisphaera shabanensis gen. nov., sp. nov., a novel,moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep,Red Sea

A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea. A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium. Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH. In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium. Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis. No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88%. The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed.     

携带新城疫病毒融合蛋白基因的重组减毒单核细胞增多性李斯特菌构建与鉴定

以鸡新城疫病毒F基因(NDV-F)为模式外源基因,通过基因切割-重叠延伸PCR法(SOE-PCR)将其插入到单核细胞增多性李斯特菌(Listeria monocytogenes)毒力基因hly的启动子和信号肽序列下游,并将该融合片段克隆入穿梭质粒pKSV7,随后将重组质粒电转李斯特菌进行同源重组。NDV-F基因的PCR扩增表明该重组菌构建成功,RT-PCR结果表明F基因在重组菌中得到了转录。比较了重组菌和野生型菌株的溶血性、黏附和侵袭力、对小鼠和鸡胚的毒力和生长特性以及重组菌的体内外稳定性,结果表明:hly基因中F片段的整合消除了单核细胞增多性李斯特菌溶血素基因的表达,其培养上清液没有溶血性,而野生型菌株的溶血价达24;细胞试验表明重组菌对细胞的黏附力和相对侵袭力均有不同程度的降低,而相对侵袭力与野生型菌株具有显著性差异(P<0.05);重组菌对小鼠及鸡胚的毒力(LD50)与野生型相比分别下降3.7和6.5个对数数量级;重组菌在BHI肉汤和小鼠体内连续5次后,仍然可以扩增出目的基因NDV-F,初步表明该重组菌较为稳定。