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PubMed2003

Coordinated down-regulation of KCNQ1 and KCNE1 expression contributes to reduction of I(Ks) in canine hypertrophied hearts.

OBJECTIVE: In animal models of hypertrophy, electrical remodeling giving rise to QT prolongation occurs rapidly and is associated with the development of torsade de pointes (TdP) arrhythmias and sudden death. Chronic AV block (CAVB)-induced hypertrophy in dogs has been associated with a reduction in the slow component (I(Ks)) of the delayed rectifier potassium current (I(K)), which contributes to a prolongation of ventricular repolarization, the development of an acquired form of long QT, and the substrate for triggered activity and TdP. The present study was designed to probe the molecular basis for the decrease in I(Ks) by studying the characteristics of KCNE1 and KCNQ1, the putative genes responsible for formation of the channel. METHODS AND RESULTS: Using a combination of Northern blot, competitive multiplex PCR and immunoblot assays, we found that CAVB reduces KCNE1 and KCNQ1 RNA in the canine ventricles by 70 and 80%, respectively. Protein levels of KCNE1 and KCNQ1 were reduced by 60 and 50%, respectively. We also demonstrate at the molecular level the basis for inter-ventricular difference in I(Ks) density previously reported in hearts of normal dogs and show the basis for reduction of this difference in the CAVB dog. CONCLUSIONS: Our results indicate that the CAVB-induced reduction in I(Ks) is due to a down-regulation of KCNE1 and KCNQ1 transcription. The data suggest that electrical remodeling of the cardiac ventricle during hypertrophy involves regulation of the gene expression through modulation of transcriptional and translational regulatory pathways. The reduction in KCNE1 and KCNQ1 expression increases the dependence of ventricular repolarization on the rapid component of I(K) and may potentiate the action of Class III antiarrhythmic agents.

Animals↗

PubMed1997

Newborn genetic identification: a protocol using microsatellite DNA as an alternative to footprinting.

Newborn identification by foot- or finger-printing presents serious drawbacks. This study proposes an alternative method based on DNA analysis of blood-spots taken from the newborn child. CSF1PO, TPOX and TH01 microsatellite loci were chosen to develop a fast and reliable protocol to be applied in cases where it is suspected that newborn children have been exchanged. The advantage of these loci is that one can simultaneously amplify them by PCR multiplex reaction and determine their alleles, thereby reducing the time needed for identification tests. Moreover, the amplification products of these loci are very small (< 350 bp) and so can be analyzed in samples with degraded DNA. We have been able to prove that it is possible to obtain results in blood-spots taken from newborns up to 13 years before and kept at room temperature. Thus the protocol proposed here can be applied in long-term post-natal identification cases.

Base Sequence↗

PubMed2001

Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue.

BACKGROUND: Deregulated c-Myc expression and alterations of c-myc oncogene have been reported to play an important role in breast cancer tumorigenesis. We examined the relationship between c-Myc protein level, amplification of c-myc oncogene and commonly used clinical and pathologic factors. METHODS: The studies were conducted on 94 ductal and lobular cancers. Amplification of c-Myc was assessed by the semiquantitative multiplex PCR assay. The amount of c-Myc protein was estimated by the densitometry analysis of Western blots. RESULTS: Amplification of c-Myc was found in 21% of examined cancers. There was no association of c-myc amplification with established risk factors. Overexpression of c-Myc protein without c-myc amplification was associated with negative status of axillary lymph node. The size of lobular carcinoma displaying overexpression of c-Myc and the normal copy number of c-myc gene was significantly smaller than the size of tumor with elevated c-Myc and amplification of c-myc gene (p < 0.01). Within tumors displaying overexpression of c-Myc protein and c-myc gene amplification the size of ductal carcinoma was smaller than the size of lobular carcinoma (p < 0.007). CONCLUSION: Data presented in this study suggest that alterations of c-myc gene and c-Myc protein level might be related to breast cancer progression. The prognostic utility of elevated level of c-Myc protein associated with normal status of c-myc gene for patients with lobular carcinoma requires further studies.

Breast↗

PubMed2001

Multiplex screening for functionally rearranged immunoglobulin variable regions reveals expression of hybridoma-specific aberrant V-genes.

Modification of antibody effector functions is commonly performed by chimerization or humanization. Cloning of antibody variable regions from hybridomas represents a first step that is frequently hampered by the expression of non-functionally rearranged variable regions in hybridoma cells that originate from MOPC21-derived fusion partners. We now present a simple method to clone functionally rearranged V-genes, based on V-gene-specific multiplex PCR screening. Using this method we document the expression of aberrant V-genes that originate from the original B-cell used for the hybridoma generation, not from the fusion partner, and are - thus - hybridoma specific.

Animals↗

PubMed2003

Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia.

Colombia is a tropical country located at the north of South America. It is considered to be one of the most important countries in terms of its biodiversity worldwide. One hundred and eight soil samples obtained from agricultural crops and wild ecosystems were evaluated in terms of the presence of Bacillus thuringiensis (Bt) native strains. One hundred and eight different Bt strains were isolated and characterized by the presence of crystal proteins by SDS-PAGE and a multiplex PCR with general and specific primers for cry1 and cry3, cry7, and cry8 gene detection. Most of the Bt strains (73%) reacted with the cry1 general primers; 27.8% of the Bt strains reacted with cry3, cry7, and cry8 general primers and 17.8% of strains did not react with any of these two sets of primers. Thirty different PCR profiles were found in the strains with cry1 genes when they were analyzed with specific primers (cry1A to cry1F). A high frequency of joint occurrence was observed for cry1Aa/cry1Ab, cry1Aa/cry1Ac, cry1Ab/cry1Ac, and cry1C/cry1D genes with a Pearson coefficient of 0.88, 0.74, 0.76, and 0.87, respectively. Other distinctive characteristics were found in the Colombian collection as the presence of 22.2% of native strains which presented, at the same time, lepidopteran and coleopteran active genes. Interesting relations were found as well between the cry gene distribution and the geographical areas sampled. Finally, some strains with moderate to high biopesticide activity against Spodoptera frugiperda (Lepidoptera) and Premnotrypes vorax (Coleoptera) insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.

Bacillus thuringiensis↗

PubMed2001

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.

Animals↗

PubMed2003

Effect of caloric restriction on Hprt lymphocyte mutation in aging rats.

Caloric restriction (CR) reduces tumor incidence and retards aging in laboratory animals, including non-human primates. Because of the relationships among mutation, disease susceptibility, and aging, we investigated whether or not CR affects the accumulation of somatic cell mutations in aging animals. Starting at approximately 2 months of age, male CD rats (Harlan Sprague-Dawley-derived) were placed on different levels of dietary intake: ad libitum (AL) feeding, and 90% (10% CR), 75% (25% CR) and 60% (40% CR) of the total calories consumed by AL animals. At 3, 6, 12, and 24 months after the beginning of CR, Hprt mutant frequencies (MFs) were determined. The MFs measured in spleen lymphocytes from AL and CR rats sacrificed at 3 months of dietary restriction were similar for all dietary groups. However, the MFs at 6, 12, and 24 months of CR were significantly higher in AL-fed rats compared with animals on 40% CR: (4.5+/-0.4)x10(-6) versus (3.3+/-0.3)x10(-6) (P=0.032) in 6 months CR rats; (10.3+/-2.3)x10(-6) versus (7.3+/-1.2)x10(-6) in 12 months CR rats (P=0.04), and (18.3+/-3.2)x10(-6) versus (7.8+/-1.0)x10(-6) (P=0.001) in 24 months CR rats. In addition, rats receiving 25% CR for 24 months had a MF, (10.7+/-2.0)x10(-6), between the 40% CR and AL rats. Multiplex PCR of the Hprt gene in mutant clones from 12 and 24 months 40% CR rats and the corresponding AL rats detected deletions in 42% of CR mutants and 19% of AL mutants. Because of the difference in Hprt MF in the two groups, the estimated MF associated with deletions in CR rats was similar to the deletion MF in AL rats. This observation implies that the lower MF in CR rats is due to a reduction in smaller Hprt mutations (i.e. base substitutions and frameshifts). The pattern of smaller Hprt mutations from AL rats suggests that many were produced by reactive oxygen species (ROS). The results indicate that CR reduces the accumulation of spontaneous somatic cell mutation in aging rats, especially those caused by base substitutions and frameshifts.

Aging↗

PubMed1998

Molecular bases of hprt mutations in malathion-treated human T-lymphocytes.

Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at the loss of one or more exons. In addition, It was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.

Humans↗

PubMed1998

Deletion-pattern analysis of alpha-particle and X-ray induced mutations at the HPRT locus of V79 Chinese hamster cells.

To investigate the mutagenic mechanisms of low-energy alpha particles V79 Chinese hamster cells were irradiated with 241Am-alpha particles (mean LET of 112 keV/micron). Parallel experiments were performed using 300 kV X-rays. Cell inactivation and mutation induction cross sections were measured. At approximately 20%--survival level, DNA deletions were analysed at the HPRT locus by multiplex-PCR-analysis of all nine exons of 47 alpha-irradiated and 36 background mutants. 92 HPRT- mutants isolated after 300 kV-X-irradiation were analysed similarly for comparison, along with 15 corresponding background mutants. The resulting mutant deletion-pattern distributions were corrected for background mutations. alpha Particles induced a larger fraction of deletions than X-rays. Furthermore, non-contiguous partial deletions were present among the alpha-induced mutants, a type not found after X-irradiation.

Alpha Particles↗

PubMed1999

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.

Anti-HIV Agents↗

PubMed1999

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators.

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991-1992 Liquidators than in Russian Controls (chi 2 = 10.5, p = 0.001) and in 1993-1994 Liquidators (chi 2 = 8.3, p = 0.004), and was marginally elevated relative to 1995-1996 Liquidators (chi 2 = 3.3, p = 0.07). This type of mutations has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991-1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p = 0.009), not with increased MF in 1991-1992 Liquidators (p = 0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p = 0.0001), and also was elevated in Liquidators sampled in 1991-1992 (72%, p = 0.0076), 1993-1994 (22%, p = 0.037), and 1995-1996 (62%, p = 0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.

Adult↗

PubMed2000

Incidence of p14ARF gene deletion in high-grade adult and pediatric astrocytomas.

The INK4a-ARF locus encodes 2 separate proteins through differential splicing of alternative first exons to produce p16INK4a (exon 1alpha) and p14ARF (exon 1beta) products in human cells. The p16INK4a protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product can complex with and sequester the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. Loss of p16INK4a expression would disrupt the retinoblastoma (Rb)/p16INK4a/cyclin D-dependent kinase (CDK4) pathway, whereas loss of p14ARF expression would inactivate both the Rb and p53/ MDM2/p14ARF pathways through MDM2, which can complex with either Rb or p53. Loss of the p16INK4a gene on 9p21 has been documented in a wide range of human tumors, including one third of glioblastomas. However, in tumors showing homozygous loss of exon 2 of the p16INK4a gene, loss of exon 1beta of the p14ARF gene has not been established. In this study, we have assessed deletion of the p14ARF gene in 29 pediatric and 107 adult high-grade astrocytomas and 9 glioma cell lines, using multiplex PCR analysis for exon 1beta. We found homozygous deletions for exon 1alpha and exon 1beta in 3 of 29 (10%) of the pediatric cases (2 grade III, 1 grade IV), 25 of 107 (23%) of the adult cases (6 grade III and 19 grade IV), and 8 of 9 (89%) of the glioma cell lines. Therefore, loss of the INK4a-ARF locus in high-grade astrocytomas may contribute to the highly malignant behavior and treatment resistance of these tumors through elimination of multiple checkpoint cell cycle control proteins.

Adult↗

PubMed2004

Sex ratios in mule duck embryos at various stages of incubation.

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.

Animals↗

PubMedDate not supplied

Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease.

The alpha-synuclein gene (SNCA) has been implicated in autosomal dominant forms of Parkinson's disease. We screened 119 individuals from families with this rare form of the disease for SNCA duplications by semiquantitative multiplex PCR. Two patients had duplications, which were confirmed by analysis of intragenic and flanking microsatellite markers. The phenotype in both patients was indistinguishable from idiopathic Parkinson's disease and no atypical features were present, by contrast with reports of families with triplication of the same gene. These results indicate that SNCA is more frequently associated with familial Parkinson's disease than previously thought, and that there is a clear dosage effect according to the number of supernumerary copies of this gene.

Adult↗

PubMed2002

Increased risk for therapy-associated hematologic malignancies in patients with carcinoma of the breast and combined homozygous gene deletions of glutathione transferases M1 and T1.

The most serious long-term complications of anti-tumor therapy are secondary malignancies. Parameters which might allow an estimation of the individual risk to develop a therapy-induced neoplasia are urgently needed. We examined whether the genotypes of the glutathione S-transferases (GST) M1 and T1, which metabolize various cytostatic drugs, as well as reactive oxygen species, influence the risk for secondary neoplasia. In a retrospective study, we analyzed peripheral blood lymphocyte or bone marrow DNA samples from 213 patients with acute myeloid leukemia (AML) and 128 with myelodysplastic syndromes (MDS) 44 of whom suffered from therapy-associated AML/MDS. The control group consisted of 239 healthy individuals with comparable composition as to race and sex. GSTM1 and GSTT1 were analyzed by multiplex PCR. Comparison between patients and control group revealed a significant (P=0.0003) overrepresentation of combined deletions of both GSTM1 and GSTT1 (double null genotype) in the group of patients with AML/MDS secondary to chemo- and/or radiotherapy of a carcinoma of the breast. In this group, 55% of the patients displayed the double null genotype as compared with 8.8% in the control group. We conclude that patients with carcinoma of the breast and inheritance of a combined gene deletion of GSTM1 and GSTT1 might bear an increased risk to develop a secondary therapy-induced hematologic neoplasia. An insufficient detoxification of cytostatic drugs such as cyclophosphamide is suggested to represent the underlying pathomechanism.

Adult↗

PubMed1999

Chronic myeloid leukemia with expression of ALL-type BCR/ABL transcript: a case-report and review of the literature.

CML with exclusive expression of ALL-type bcr/abl has only been rarely described. In some cases, the presence of this fusion gene has been associated to a differentiated subtype of CML that share some features with CMML, while in another case this molecular hallmark has been associated to a bad prognosis of the disease with a blast phase as clinical presentation or an early transformation to blast phase. We report a case of a 30-year-old woman who was diagnosed of CML in chronic phase in May 1989. She received treatment first with busulfan, achieving hematological remission and afterwards with interferon and Hydroxiurea. In February 1998, she was admitted at our hospital for an ABSCT. Then, molecular studies were performed. Multiplex PCR revealed the presence of a 481 bp product identified as the ela2 bcr/abl transcript and confirmed by sequencing. After 9 years from diagnosis, the patient remains in hematological remission and in good clinical condition.

Adult↗

PubMed1997

Identification and characterization of polymorphisms in the promoter region of the human Apo-1/Fas (CD95) gene.

Apo-1/Fas (CD95) is a transmembrane protein expressed on the cell surface that is involved in apoptosis and plays an important role in the function and regulation of the immune system. Aberrant expression of the Apo-1/Fas gene product has been reported in a number of immune-related disorders, such as autoimmune lymphoproliferative syndrome and systemic lupus erythematosus in humans. Mutations in the coding sequence of the Apo-1/Fas gene have been reported in the former condition, whereas no abnormalities of the gene have been found to account for the increased gene expression noted in SLE. We screened the whole 5' flanking region of the Apo-1/Fas gene encompassing over 2000 bp for mutation(s)/polymorphism(s) using multiplex PCR, single-strand conformation polymorphism (SSCP) analysis and sequencing techniques, and identified two polymorphisms in this region. The first polymorphism is a CG-->CA substitution at -1377 nucleotide position within the silencer region, which neither creates or deletes any restriction enzyme sites but alters the transcription factor SP-1 binding site. This polymorphism is noted in 20% of normal Caucasians. The second polymorphism is an GA-->GG substitution at -670 nucleotide position in the enhancer region that creates a MvaI restriction fragment length polymorphism (RFLP) and abolishes the binding site of nuclear transcription element GAS. The MvaI RFLP is polymorphic with heterozygosity of 52% and the frequency of G and A alleles are 0.49 and 0.51, respectively. The identification and characterisation of these two new polymorphisms, particularly the MvaI RFLP marker, provides new genetic markers and may prove useful for further studies on the regulation of apoptosis mediated by the Apo-1/Fas gene on human chromosome 10q23.

Apoptosis↗

PubMed2002

Development of a model meat system and investigation of the growth characteristics and genetic stability of Escherichia coli O157:H7, in the absence of meat microflora.

Model meat systems were produced using both aseptically procured and irradiated raw minced beef to initially compare the growth characteristics of a three-strain mixture of Escherichia coli O157:H7 in each medium. A multiplex PCR assay (detecting VT1, VT2 and eae genes) was used to determine the proportion of individual strains recovered at each sampling time by virtue of the different combinations of these virulence factors encoded by each strain, and to investigate their genetic stability. No differences in the growth characteristics of the pathogen (P>0.05) were recorded in the meat matrices, irrespective of the preparation method, thus validating the use of irradiation to sterilise (42 kGy, in vacuo at <-5 degrees C) minced meat in the production of control meat matrices for application to further research investigations. A novel plating method, incorporating a period of catalase-induced resuscitation, was found to give significantly higher recovery of the pathogen (P<0.05) from these meat matrices when compared to conventional spread-plating on Trypticase Soy Agar (TSA). A proportion of the recovered populations, however, was found to be failing to produce an amplicon relating to a Verotoxin (VT) gene. A further investigation demonstrated that O157:H7 strain E 90197 (VT2 and eae positive) was displaying genetic instability during growth in and recovery from meat matrices with the apparent loss of the VT2 gene. An alternative VT2 and eae positive strain (ATCC 43889) demonstrated no such genetic instability, demonstrating inter-strain variation for this phenomenon. Thus, it should be considered that an essential pre-requisite to studies relying upon the maintenance of the pathogenic potential of E. coli O157:H7 should be an evaluation of the genetic stability of proposed strains of this serotype.

Animals↗