A simple PCR-based genotyping method for M105I mutation of alpha-SNAP enhances the study of early pathological changes in hyh phenotype

α-SNAP is an essential component of the protein machinery responsible for membrane fusion events in different cell types. The hyh (hydrocephalus with hop gait) mouse carries a missense mutation in Napa gene that results in a point mutation (M105I) in α-SNAP protein. Homozygous animals for the mutant allele have been identified by the clinical and/or neuropathological phenotype, or by direct sequencing of PCR products. The aims of the present study were (i) to develop a high-throughput technique to genotype hyh mice, (ii) to correlate genotype-phenotype, and (iii) to analyze the earliest pathological changes of hyh mutant mice. As no restriction sites are affected by the hyh mutation, we resolved this problem by creating a BspHI restriction site with a modified (mismatch) polymerase chain reaction (PCR) primer in wild-type allele. This artificially created restriction site (ACRS)–PCR technique is a simple, rapid and reliable method to genotype hyh mice in a day-work procedure. Biochemical and histological analysis of genotyped hyh embryos at different developmental stages allowed us to identify and characterize the earliest brain pathological changes of the hyh phenotype, including the first signs of neuroepithelial disruption and neuronal ectopia. In addition, genotype-phenotype analysis of 327 animals confirmed that (i) hyh is a single-gene autosomal recessive disorder, and (ii) the disorder has 100% penetrance (i.e., the mutation was only present in affected mice). The genotyping method described here enhances the potentiality of hyh mouse as a unique in vivo model to study the role of membrane trafficking in different developmental and physiological processes.     

Do in situ forming PLG/NMP implants behave similar in vitro and in vivo? A non-invasive and quantitative EPR investigation on the mechanisms of the implant formation process

Electron paramagnetic resonance (EPR) spectroscopy was applied to monitor non-invasively the formation of in situ forming implants in vitro and in vivo after the administration of poly(lactide-co-glycolide) (PLGA)/N-methyl-pyrrolidone (NMP) solutions. The nitroxide spin probe 4-benzoyloxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TB) was incorporated in polymer solutions and samples were incubated in 0.1 M phosphate buffer (pH 7.4) at 37 °C or injected subcutaneously in the femoral of BALB/c mice. EPR permitted the direct and continuous determination of the NMP-water exchange during implant formation both in vitro and in living mice. The formation of the implant structure followed a two phase mechanism: over 75% of the polymer precipitated immediately after injection within the first 30 min and formed a solid shell. The subsequent moderate solidification of the implants was governed by diffusion and was completed after 24 h. The replacement of the organic solvent NMP by water was determined by polarity shifts within the implant and could be quantified. Both the kinetic of NMP-water exchange and polymer precipitation showed good in vitro–in vivo correlation.     

A new model of rectal cancer with regional lymph node metastasis allowing in vivo evaluation by imaging biomarkers

Object

The work is aimed to develop a murine model of rectal cancer, which could be used to monitor lymph node metastasis development by magnetic resonance imaging (MRI) and optical imaging (OI) techniques.

Subjects and methods

Ht-29 cancer cells were directly injected into the submucosal layer of the rectum of athymic nude mice using trans-anal rectal cancer cell injection (TARCI). Thirty-six mice were inoculated with 10 × 10⁵ cells and five mice were treated with sterile phosphate buffer solution. One to 4 weeks after cell injection, tumor growth was evaluated in vivo using T2-weighted MRI at 4.7T. A further group of animal (n = 6) treated with ht-29_luc cells, with the same protocol, was monitored by optical imaging. In both groups, the presence of the primary tumor and of lymph nodes metastasis was confirmed by histology.

Results

In all animals, primary tumors were detectable by MRI, 1 week from TARCI. After 4 weeks primary tumors showed a mean longitudinal diameter of about 2 cm. All animals developed regional lymph node metastases. Others organs (e.g. lung or liver) were not affected. In fat-suppressed, T2-weighted MRI, lymph nodes appeared as small areas characterized by hyper-intense signal compared to muscle. OI permitted evaluation of the primary tumor growth in perineal region.

Conclusions

TARCI of ht-29 cells into the rectum of nude mice is a feasible way to obtain a easily reproducible model of regional lymph node metastases could be monitored by magnetic resonance and optical imaging techniques.     

High prevalence of erythromycin resistance and macrolide-resistance genes, mefA and ermB , in Streptococcus pneumoniae isolates from the upper respiratory tracts of children in the Sapporo district, Japan

Our previous study demonstrated that the frequency of penicillin-resistant Streptococcus pneumoniae (PRSP) was lower in our district than in districts in other Japanese studies. In this study, we investigated the prevalence of erythromycin resistance. The susceptibility to erythromycin and the distribution of the macrolide-resistance genes, mefA and ermB, were examined in S. pneumoniae isolates from the upper respiratory tracts of children in four cities in the Sapporo district, Hokkaido prefecture, Japan. Of the 156 isolates, 27 (17.3%) were erythromycin-sensitive, 6 (3.9%) were erythromycin-intermediately resistant, and 123 (78.9%) were erythromycin-resistant. Fifty-nine (37.8%) had the mefA gene, 89 (57.1%) had the ermB gene, and 129 (82.7%) had the mefA and/or the ermB gene. The ermB-positive isolates tended to show high resistance to erythromycin. Erythromycin-resistant isolates and the macrolide-resistance genes were often present in infants or younger children. The frequency of erythromycin-resistant isolates in the four cities was very high, ranging from 76.3% to 83. 3%, as high as the national average. Although erythromycin-resistant isolates generally tend to show cross-resistance to penicillin, the frequency of PRSP was very low in this study, as compared with other Japanese studies. Erythromycin resistance was frequently recognized not only in PRSP but also in penicillin-sensitive S. pneumoniae (PSSP) as well. In Japan, erythromycin resistance may have already become widespread, even in local areas where penicillin resistance is not especially prevalent. A. Harimaya and S. Yokota contributed equally to this work.     

Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase blaCMY-4 promoted by insertion sequence ISEcp1

A Klebsiella pneumoniae strain, KU6500, which showed resistance to extended-spectrum β-lactams and produced the plasmid-encoded AmpC β-lactamase CMY-4, was identified from clinical isolates in Japan. The aim of this study was to identify the mechanism of the high-level expression of bla _CMY-4. Sequence analysis indicated that the promoter element of Citrobacter freundii was conserved, but the insertion sequence ISEcp1 coding with the putative promoter element, was inserted into the AmpR binding site. We determined the influence of the promoter on bla _CMY-4 expression and β-lactam resistance. Two recombinant plasmids containing the entire bla _CMY-4 gene, with or without the ISEcp1-mediated promoter sequences, were constructed and named pMWampC and pMWISEcp1, respectively. Escherichia coli DH5α (pMWISEcp1) was resistant to almost all β-lactams tested and E. coli DH5α (pMWampC) was susceptible to all, except for cephalothin. In addition, the activity of each promoter was measured by subcloning the element into a promoterless luciferase plasmid pGL3-Basic vector. The expression of the putative promoter of ISEcp1 was 18.9-fold higher than that of C. freundii. These results suggest that the putative promoter element of ISEcp1 is necessary for the high-level expression of bla _CMY-4 to confer resistance to extended-spectrum cephalosporins.     

The AraC-family regulator GadX enhances multidrug resistance in Escherichia coli by activating expression of mdtEF multidrug efflux genes

Multidrug efflux pumps contribute to the resistance of Escherichia coli to many antibiotics and biocides. Here, we report that the AraC-family regulator GadX increases multidrug resistance in E. coli through activation of the MdtEF efflux pump. Screening of random fragments of genomic DNA for ability to increase β-lactam resistance led to the isolation of a plasmid containing gadX, which codes for the regulator of acid resistance. When overexpressed, gadX significantly increased the resistance of the E. coli strain to oxacillin, cloxacillin, nafcillin, erythromycin, rhodamine 6G, and sodium dodecyl sulfate. The increase in drug resistance caused by gadX overexpression was completely suppressed by deleting the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Quantitative real-time polymerase chain reaction (PCR) showed that GadX activated the expression of mdtEF but none of the other drug efflux pumps in E. coli. Deletion of mdtEF completely suppressed GadX-mediated multidrug resistance. Our results indicate that the GadX regulator, in addition to its role in acid resistance, increases multidrug resistance in E. coli by activating the MdtEF multidrug efflux pump.     

Comparison of phenotypic and genotypic antimicrobial profiles in Escherichia coli and Salmonella enterica from the same dairy cattle farms

Transmission of antimicrobial drug resistance from resistant bacteria to non-resistant strains is an important public health issue. In this study, we have examined the possibility of multiple resistance gene transfer between Escherichia coli and Salmonella in the natural setting. Bacteria isolated from calves concurrently shedding E. coli and Salmonella showed similar antimicrobial drug resistance patterns as measured by a broth dilution method. However, microarray analysis of the antibiotic resistance at the gene level revealed several differences in resistance gene profile. Resistance profiles of E. coli isolated from different farms were closer than the profile of E. coli and Salmonella isolated from the same farm. This shows that the chance of multiple resistance gene transfers between these species is unlikely.     

PEGylation improves pharmacokinetic profile, liver uptake and efficacy of Interferon gamma in liver fibrosis

Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20 kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled ¹²⁵I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl₄-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases.     

Feasibility,sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.     

In vitro activities of new ketolide, telithromycin, and eight other macrolide antibiotics against Streptococcus pneumoniae having mefA and ermB genes that mediate macrolide resistance

The comparative in vitro activity of a new ketolide, telithromycin (TEL), and eight other macrolide-lincosamide antibiotics (MLS) against 215 strains, of Streptococcus pneumoniae including penicillin-resistant isolates (PRSP), was determined by the agar dilution method. These strains were isolated from patients with pneumonia, otitis media, and purulent meningitis between 1995 and 1997. Two genes, mefA and ermB, that encode MLS resistance in the strains were identified by polymerase chain reaction (PCR). Of the strains, 30.2% (n = 65) had the mefA gene, 37.7% (n = 81) had the ermB gene, and 1.4% (n = 3) had both resistant genes. The minimum inhibitory concentration (MIC_90s) of TEL and 16-membered ring MLS for strains having the mefA gene were 0.063–0.25µg/ml, which were the same level as those for MLS-susceptible strains. On the other hand, the strains with the mefA gene showed low-level resistance to 14- and 15-membered ring MLS, with MIC_90s ranging from 1 to 4µg/ml. Only the MIC₉₀ of TEL at 0.5µg/ml, for strains having the ermB gene was superior to that of the 14-, 15-, and 16-membered ring MLS (MIC₉₀, 64µg/ml). TEL also showed excellent activity against PRSP having abnormal pbp1a, pbp2x, and pbp2b genes. Most strains having the mefA and ermB genes were serotyped to 3, 6, 14, 19, and 23. These results suggest that TEL may be a useful chemotherapeutic agent for respiratory tract infections caused by S. pneumoniae.     

The property of methylated APC gene promotor and its influence on lung cancer cell line

Previous findings have suggested that methylation of the APC gene may be associated with some tumors including lung cancer. To explore the pattern of APC methylation and the effect of APC gene methylation on its protein expression in lung cancer cell lines, we investigated APC promotor methylation by methylation specific PCR (MSP) and bisulfite sequencing and analyzed the APC protein levels by western blot in three lung cancer cell lines. Monoallelic methylation and 20 methylated CpGs in CpG island near the open reading frame (ORF) of the APC gene were found in the NCI-H460 cell line, and were stablely inherited within 10 generations of the cell line in culture. Our results showed that two special CpG sites (794, 797) might be binding sites for proteins that regulate APC expression. Protein expression of the APC gene in the NCI-H460 cell line declined, but was enhanced after the treatment with 5-aza-2-deoxycytidine (5-aza-dC). Inherited monoallelic methylation of the APC gene may play an important role in lung cancer. Demethylation of the APC gene by 5-aza-dC may be useful for the treatment of lung cancer.     

Conditional Bicistronic Cre Reporter Line Expressing Both Firefly Luciferase and β-galactosidase

Purpose  

The Cre-loxP system has become an important strategy for conditional gene deletion and conditional gene expression in genetically engineered mice. To evaluate Cre recombinase expression, we generated reporter mice that permit both noninvasive imaging in living animals and either ex vivo histochemical/immunohistochemical tissue transgene expression analysis or quantitative enzyme analysis in the same animal.     

MRI影像组学预测结直肠癌患者KRAS基因突变

目的 构建MR T2WI影像组学模型,评价其预测结直肠癌患者Kirsten大鼠肉瘤（KRAS）病毒癌基因亚型的价值。方法 将99例经病理证实的结直肠癌患者分为训练组（n=68）及验证组（n=31）,根据KRAS基因检测结果进一步将其分为突变亚组及野生亚组,训练组2亚组分别含36、32例,验证组2亚组分别含16、15例,比较亚组间实验室检查结果及肿瘤大小的差异;提取并筛选训练组MR T2WI影像组学特征,构建影像组学模型、临床模型及影像组学-临床联合模型。绘制受试者工作特征（ROC）曲线,计算曲线下面积（AUC）,评价各模型预测结直肠癌患者KRAS基因亚型的效能;以DeLong检验比较各模型间效能差异。通过校正曲线分析3种模型的校正性能,以Hosmer-Lemeshow检验评价校准曲线的校准度;以决策曲线分析（DCA）评价3种模型临床应用价值。结果 训练组和验证组内亚组间实验室检查结果及肿瘤大小差异均无统计学意义（P均>0.05）。共提取3个组学特征用于构建预测模型。影像组学模型与临床模型、影像组学-临床联合模型预测2组KRAS基因亚型的AUC差异均无统计学意义（P均>0.05）;影像组学-临床联合模型预测训练组KRAS基因亚型的AUC显著高于临床模型（P<0.05）,但在验证组差异均无统计学意义（P>0.05）。校准曲线及Hosmer-Lemeshow检验显示3种模型预测值和观察值的一致性良好（P均>0.05）。影像组学模型和影像组学-临床联合模型在2组中的DCA曲线净收益值均高于临床模型。结论 MR T2WI影像组学纹理特征预测结直肠癌患者KRAS基因突变亚型具有一定潜力。     

PiggyBac Transposon-based Inducible Gene Expression In Vivo After Somatic Cell Gene Transfer

Somatic cell gene transfer has permitted inducible gene expression in vivo through coinfection of multiple viruses. We hypothesized that the highly efficient plasmid-based piggyBac transposon system would enable long-term inducible gene expression in mice in vivo. We used a multiple-transposon delivery strategy to create a tetracycline-inducible expression system in vitro in human cells by delivering the two genes on separate transposons for inducible reporter gene expression along with a separate selectable transposon marker. Evaluation of stable cell lines revealed 100% of selected clones exhibited inducible expression via stable expression from three separate transposons simultaneously. We next tested and found that piggyBac-mediated gene transfer to liver or lung could achieve stable reporter gene expression in mice in vivo in either immunocompetent or immune deficient animals. A single injection of piggyBac transposons could achieve long-term inducible gene expression in the livers of mice in vivo, confirming our multiple-transposon strategy used in cultured cells. The plasmid-based piggyBac transposon system enables constitutive or inducible gene expression in vivo for potential therapeutic and biological applications without using viral vectors.     

Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H, K-ATPase

We purified a compound with strong inhibitory effect on H⁺, K⁺-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-β- -glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC₅₀ of the compound for H⁺, K⁺-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K⁺. Pentagalloylglucose had relatively weak inhibitory effects for Mg⁺-ATPase (IC₅₀: >10 μmol/l) and Na⁺, K⁺-ATPase (IC₅₀: 2.7 μmol/l). Pentagalloylglucose also inhibited the accumulation of [¹⁴C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 μmol/l histamine (IC₅₀: 7.8 μmol/l) and 1 mmol/l dbc-AMP (IC₅₀: 10 μmol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H⁺, K⁺-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

The evaluation of putative endogenous control housekeeping genes for real-time polymerase chain reaction expression studies in Moraxella catarrhalis

Comparisons of endogenous control genes in real-time polymerase chain reaction gene expression studies involving Moraxella catarrhalis are rare. This study shows that a combination of the iron sequestering gene copB and 16S rRNA genes would be useful for lineage 1 (16S rRNA type 1) isolates, but not lineage 2 (16S rRNA types 2 and 3) isolates.     

In vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma

Edelfosine is the prototype molecule of a family of anticancer drugs collectively known as synthetic alkyl-lysophospholipids. This drug holds promise as a selective antitumor agent, and a number of preclinical assays are in progress. In this study, we observe the accumulation of edelfosine in brain tissue after its oral administration in Compritol® and Precirol® lipid nanoparticles (LN). The high accumulation of edelfosine in brain was due to the inhibition of P-glycoprotein by Tween® 80, as verified using a P-glycoprotein drug interaction assay. Moreover, these LN were tested in vitro against the C6 glioma cell line, which was later employed to establish an in vivo xenograft mouse model of glioma. In vitro studies revealed that edelfosine-loaded LN induced an antiproliferative effect in C6 glioma cell line. In addition, in vivo oral administration of drug-loaded LN in NMRI nude mice bearing a C6 glioma xenograft tumor induced a highly significant reduction in tumor growth (p < 0.01) 14 days after the beginning of the treatment. Our results showed that Tween® 80 coated Compritol® and Precirol® LN can effectively inhibit the growth of C6 glioma cells in vitro and suggest that edelfosine-loaded LN represent an attractive option for the enhancement of antitumor activity on brain tumors in vivo.     

Comparison of [<Superscript>14</Superscript>C]FMAU, [<Superscript>3</Superscript>H]FEAU, [<Superscript>14</Superscript>C]FIAU,and [<Superscript>3</Superscript>H]PCV for Monitoring Reporter Gene Expression of Wild Type and Mutant Herpes Simplex Virus Type 1 Thymidine Kinase in Cell Culture

Purpose To assess the optimal reporter probe/reporter gene combination for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression, we compared the cellular uptake of 1-(2′-fluoro-2′-deoxy-d-arabinofuranosyl)-5-methyluracil (FMAU), 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (FEAU), 2′-fluoro-2′-deoxy-β-d-arabinofuranosyl-5-iodouracil (FIAU) and penciclovir (PCV) in both HSV1-tk and HSV1-sr39tk expressing cells. Procedures For stably transfected cell studies, C6 rat glioma cells, C6 HSV1-tk transfectant, C6 mutant HSV1-sr39tk transfectant, rat Morris hepatoma cells (MH3924A), and MH3924A HSV1-tk transfectant cells were used. For adenoviral infection studies, C6 rat glioma cells were exposed to serial titers of AdCMV–HSV1-tk, AdCMV–HSV1-sr39tk, or AdCMV–fluc for 24 hours. These cells were incubated with [¹⁴C]FMAU, [³H]FEAU, [¹⁴C]FIAU, and [³H]PCV, and cellular uptake of radioactivity was measured. Results [³H]FEAU exhibited the highest or second highest accumulation and the most selectivity regardless of the mode of gene transfer for both HSV1-tk and mutant HSV1-sr39tk reporter genes. Conclusion This combination of high accumulation and high selectivity for both HSV1-tk and HSV1-sr39tk makes suitably radiolabeled FEAU a promising candidate as a radiotracer for imaging HSV1-tk/HSV1-sr39tk gene expression in living subjects.     

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3 · 3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10⁴–10⁵ by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.     

The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli

The presence of the enteroaggregative Escherichia coli (EAEC) virulence genes aatA, aap, and aggR was assayed in strains of different diarrheagenic E. coli pathotypes and nonpathogenic E. coli. The dispersin-encoding gene (aap) was detected in EAEC, diffusely adherent E. coli, and nonpathogenic E. coli, demonstrating that molecular diagnostics of EAEC based on aap detection may identify non-EAEC strains.