Antibiotic sensitivity of Staphylococcus aureus strains isolated from fishery products

During the course of an investigation on the incidence of Staphylococcus aureus contamination in frozen crab meat, frozen prawns and dried fishes from Cochin, 116 strains of S. aureus were isolated. The sensitivity of the isolated strains towards nine antibiotics showed that all the S. aureus strains were sensitive to kanamycin and streptomycin (100%). Sensitivity to other antibiotics like chloramphenicol, polymyxin-B, erythromycin, tetracycline, neomycin, penicillin and ampicillin were shown by 98.28, 93.10, 87.93, 68.10, 67.24, 56.90 and 55.17 percent of the isolates respectively.

Antibiotic sensitivity of Vibrio parahaemolyticus strains

The strains of Vibrio parahaemolyticus isolated from water, sediment, plankton, fish and prawn of Cochin backwater were tested for sensitivity to ten antibiotics namely, ampicillin, chloramphenicol, gentamycin, kanamycin, neomycin, oxytetracycline, penicillin, polymyxin-B, streptomycin and sulphadiazine. Of the 120 isolates tested, 96.7 and 93.3% were sensitive to gentamycin and chloramphenicol respectively. No strain was sensitive to penicillin and only 5% were sensitive to kanamycin. Isolates from fish and prawn showed higher resistance to ampicillin and none of them was sensitive to kanamycin. Multiple resistant V. parahaemolyticus strains were more in prawn than in other samples.

Antibiotic resistance of Staphylococcus aureus strains isolated from fish processing factory workers

One hundred and twenty two strains of Staphylococcus aureus isolated from throats and palms of 39 workers from 6 fish processing factories situated in and around Cochin were tested for their sensitivity to nine commonly used antibiotics-ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, penicillin, polymyxin-B, streptomycin and tetracycline. Highest percentage of resistance was observed towards ampicillin followed by penicillin i.e. 64.75% and 59.84%. Resistance towards other antibiotics like tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin were shown by 22.95, 16.39, 7.38, 5.74, 3.28 and 1.64% of the isolates respectively.

Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chIL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chIL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.

Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

细菌二氯甲烷脱氯酶基因对拟南芥的转化

The dichloromethane dehalogenase gene (dcm) of Methylophilus was cloned into the plasmid pKYLX71-35S2 and fused with the 35S2 promotor,then transformed Arabidopsis by the infiltration mediated with Agrobacterium tumefaciens. Homozygous dcm seeds were obtained after several generations selection on the medium with 50 mg L -1 kanamycin.Northern blotting showed dcm mRNA was high in cytoplasm and the detection of DCM enzyme indicated that the lines containing high dcm mRNA expressed high DCM enzyme activities. Fig 4, Ref 9

Genetic mechanisms of multi-antimicrobial resistance in a pathogenic Edwardsiella tarda strain

TX01, a pathogenic Edwardsiella tarda strain isolated from diseased fish at an epidemic-inflicted fish farm in China, exhibits resistance to multiple classes of antimicrobial agents. The genes (kn(R). catA3, and tet(A), respectively) encoding resistance to kanamycin, chloramphenicol, and tetracycline were cloned and found to be 99-100% identical to the corresponding genes carried by known plasmids and transposons of human, animal, and environmental isolates. Further study demonstrated that TX01 harbors a plasmid, pETX, which proved to be (i) the carrier of the tet and cut operons; (ii) a mobile genetic element that is capable of transferring between bacteria of different genera. These results, which, to our knowledge, documented for the first time the co-existence of chloramphenicol and tetracycline resistance determinants on a conjugative plasmid in a pathogenic E tarda strain, indicated that gene acquisition via horizontal transferring of pETX-like mobile genetic entities may have played an important part in the dissemination of antimicrobial resistance and that there have existed for some time widespread genetic exchanges between bacteria of human, animal/fish, and environmental origins. (C) 2008 Elsevier B.V. All rights reserved.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens that cause severe lung infections in patients with cystic fibrosis and display extreme intrinsic resistance to antibiotics including antimicrobial peptides. B. cenocepacia BCAL2157 encodes a protein homologous to SuhB, an inositol-1-monophosphatase from Escherichia coli, which was suggested to participate in posttranscriptional control of gene expression. In this work we show that a deletion of the suhB-like gene in B. cenocepacia (?suhBBc) was associated with pleiotropic phenotypes. The ?suhBBc mutant had a growth defect manifested by an almost 2-fold increase in the generation time relative to the parental strain. The mutant also had a general defect in protein secretion, motility and biofilm formation. Further analysis of the Type-2 and the Type-6 secretion systems activities revealed that these secretion systems were inactive in the ?suhBBc mutant. In addition, the mutant exhibited increased susceptibility to polymyxin B but not to aminoglycosides like gentamicin and kanamycin. Together, our results demonstrate that suhBBc deletion compromises general protein secretion including the activity of T2SS and T6SS, and affects polymyxin B resistance, motility, and biofilm formation. The pleiotropic effects observed upon suhBBc deletion demonstrate that suhBBc plays a critical role in the physiology of B. cenocepacia.

Antimicrobic susceptibility and plasmid profile analysis as identity tests for multiple blood isolates of coagulase-negative staphylococci

We compared a disk diffusion antimicrobic susceptibility panel with plasmid DNA profiles as tests for identity of 106 isolates of coagulase-negative staphylococci cultured from the blood of 45 patients on multiple occasions. The antimicrobic panel included penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin. Nineteen patterns of antimicrobic susceptibility were found. The most common pattern was present in 25% of the isolates, and at least one isolate from 31% of the patients had this pattern. Forty-seven distinct plasmid DNA profiles were found. The most common plasmid profile was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Twenty-eight patients had multiple isolates that were identical by plasmid profile analysis. Twenty-seven (96%) of these patients had isolates that were also identical by antimicrobic susceptibility. Nineteen patients had multiple isolates that were different by plasmid profile analysis. In 18 (95%) of these patients, the isolates were also different by antimicrobic susceptibility. Although plasmid DNA profile analysis is a more discriminating tool, these data confirm that a selected disk diffusion antimicrobic susceptibility panel may be used to screen multiple blood isolates of coagulase-negative staphylococci for identity or differences.

Retroviral-mediated gene transfer of the human erythropoietin gene into a factor-dependent cell line, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.