بفضل قاعة ميكايلا، لاريسا بيسبو وغوستافو تافاريس للمساعدة التقنية.

<ol>
	<li>Bush, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+antibiotic+resistance.">Tackling antibiotic resistance.</a> Nature Rev. Microbiol. 9, 894-896 (2011).</li><li>Davies, J., Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+evolution+of+antibiotic+resistance.">Origins and evolution of antibiotic resistance.</a> Microbiol. Mol. Biol. Rev. 74 (3), 417-433 (2010).</li><li>Costa, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+is+ancient.">Antibiotic resistance is ancient.</a> Nature. 477 (7365), 457-461 (2011).</li><li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+consequences+of+antibiotic+use+for+the+resistome,+mobilome+and+microbial+pangenome.">Evolutionary consequences of antibiotic use for the resistome, mobilome and microbial pangenome.</a> Front. Microbiol. 4, 4 (2013).</li><li>Gillings, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+class+1+integrons+and+the+rise+of+antibiotic+resistance.">The evolution of class 1 integrons and the rise of antibiotic resistance.</a> J. Bacteriol. 190 (14), 5095-5100 (2008).</li><li>Brassard, S., Lapointe, J., Roy, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+relative+strength+of+tandem+promoters+for+the+antibiotic-resistance+genes+of+several+integrons.">Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons.</a> Gene. 142 (1), 49-54 (1994).</li><li>Partridge, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+the+attI1+site+of+class+1+integrons.">Definition of the attI1 site of class 1 integrons.</a> Microbiol. 146 (11), 2855-2864 (2000).</li><li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrons:+Past,+Present,+and+Future.">Integrons: Past, Present, and Future.</a> Microbiol. Mol. Biol. Rev. 78 (2), 257-277 (2014).</li><li>Partridge, S. R., Tsafnat, G., Coiera, E., Iredell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+cassettes+and+cassette+arrays+in+mobile+resistance+integrons.">Gene cassettes and cassette arrays in mobile resistance integrons.</a> FEMS Microbiol. Rev. 33 (4), 757-784 (2009).</li><li>Gillings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+class+1+integron-integrase+gene+as+a+proxy+for+anthropogenic+pollution.">Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution.</a> ISME J. In press, (2014).</li><li>Gaze, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+anthropogenic+activity+on+the+ecology+of+class+1+integrons+and+integron-associated+genes+in+the+environment.">Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment.</a> ISME J. 5, 1253-1261 (2011).</li><li>Gerzova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microbiota+composition+and+presence+of+selected+antibiotic+resistance+genes+in+carriage+water+of+ornamental+fish.">Characterization of microbiota composition and presence of selected antibiotic resistance genes in carriage water of ornamental fish.</a> PLoS ONE. 9, e103865 (2014).</li><li>Power, M., Emery, S., Gillings, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+the+wild:+dissemination+of+antibiotic+resistance+determinants+via+a+species+recovery+program.">Into the wild: dissemination of antibiotic resistance determinants via a species recovery program.</a> PLoS ONE. 8, e63017 (2013).</li><li>Stokes, H. W., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+flow,+mobile+genetic+elements+and+the+recruitment+of+antibiotic+resistance+genes+into+Gram-negative+pathogens.">Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens.</a> FEMS Microbiol. Rev. 35 (5), 790-819 (2011).</li><li>Moura, A., Oliveira, C., Henriques, I., Smalla, K., Correia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+diversity+of+conjugative+plasmids+in+integron-carrying+bacteria+from+wastewater+environments.">Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments.</a> FEMS Microbiol. Lett. 330 (2), 157-164 (2012).</li><li>Schl&#252;ter, A., Krause, L., Szczepanowski, R., Goesmann, A., P&#252;hler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+and+composition+of+a+plasmid+metagenome+from+a+wastewater+treatment+plant.">Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant.</a> J. Biotech. 136 (1-2), 65-76 (2008).</li><li>Taylor, N. G. H., Verner-Jeffreys, D. W., Baker-Austin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+systems:+maintaining,+mixing+and+mobilising+antimicrobial+resistance.">Aquatic systems: maintaining, mixing and mobilising antimicrobial resistance.</a> TREE. 26 (6), 278-284 (2011).</li><li>Stalder, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+impact+of+hospital+effluent+on+dissemination+of+the+integron+pool.">Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool.</a> ISME J. 8, 768-777 (2014).</li><li>Allen, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Call+of+the+wild:+antibiotic+resistance+genes+in+natural+environments.">Call of the wild: antibiotic resistance genes in natural environments.</a> Nature Rev. Microbiol. 8, 251-259 (2010).</li><li>Wellington, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+natural+environment+in+the+emergence+of+antibiotic+resistance+in+Gram-negative+bacteria.">The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria.</a> Lancet Infect. Dis. 13 (2), 155-165 (2013).</li><li>Gillings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+a+Tn402-like+class+1+integron+with+a+novel+cassette+array+via+flanking+miniature+inverted-repeat+transposable+element-like+structures.">Mobilization of a Tn402-like class 1 integron with a novel cassette array via flanking miniature inverted-repeat transposable element-like structures.</a> Appl. Env. Microbiol. 75 (18), 6002-6004 (2009).</li><li>Graham, D. W., Collignon, P., Davies, J., Larsson, D. J., Snape, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underappreciated+role+of+regionally+poor+water+quality+on+globally+increasing+antibiotic+resistance.">Underappreciated role of regionally poor water quality on globally increasing antibiotic resistance.</a> Env. Sci. Technol. 48 (20), 11746-11747 (2014).</li><li>Perry, J. A., Westman, E. L., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistome:+what's+new.">The antibiotic resistome: what's new.</a> Curr. Opinion Microbiol. 21, 45-50 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J. Vis. Exp. 63, e3998-e3998 (2011).</li><li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples.">Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples.</a> Env. Microbiol. 1096, 17-23 (2014).</li><li>Sajjad, A., Holley, M. P., Labbate, M., Stokes, H., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module.">Preclinical class 1 integron with a complete Tn402-like transposition module.</a> Appl. Env. Microbiol. 77 (1), 335-337 (2011).</li><li>Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

الكتاب ليس لديهم ما ذات الصلة في الكشف عنها.

تحديد integrons وأشرطة الجينات المرتبطة بها قد يمثل خطوة رئيسية في توقع ظهور مسببات الأمراض الانتهازية الجديدة، وتتبع مسارات لمسببات الأمراض في السلسلة الغذائية للإنسان، وتحديد مقاومة جديدة والفوعة محددات 8،21،26. وكان الهدف من هذه الورقة لوصف نهج مبسط لفحص عينات ل...

كان اكتشاف المضادات الحيوية واحدة من أعظم الإنجازات العلمية في القرن التاسع 20. ومع ذلك، فقد أدى استخدام وإساءة استخدام المضادات الحيوية لتطور السريع للبكتيريا المقاومة للمضادات الحيوية، وهذه تشكل الآن تهديدا خطيرا للصحة العامة في القرن الحادي و21. صعود سلالات بكتيرية مقاومة لمعظم خيارات العلاج يزيد من احتمال أننا ندخل عصر حيث العقاقير المضادة للميكروبات لم تعد فعالة 1،2. الآلية الوراثية التي تمنح المقاومة للمضادات الحيوية هو نظام القديم، سبقت البشر والضغوط اختيار المضادات الحيوية من قبل الملايين من 3 سنوات. العناصر الجينية المحمولة، مثل البلازميدات والترانسبوزونات والجزر الجينومية والعناصر وintegrons اقترانية التكاملية يمكن نشر الجينات المقاومة للمضادات الحيوية (ARG) داخل وبين الأنواع البكتيرية 4 على حد سواء. من هذه، وقد لعبت integrons دورا مركزيا في انتشار ARG، على الرغم منحقيقة أنها تعتمد على البلازميدات والترانسبوزونات للتعبئة والإدراج في الجينوم البكتيري 5. Integrons التقاط أشرطة الجينات باستخدام integron-integrase، ومن ثم التعبير عن أشرطة باستخدام integron المشفرة المروج 6،7 (الشكل 1). Integron الجينات أشرطة عناصر المحمولة الصغيرة التي تتكون من واحد إطارات القراءة المفتوحة (ORF) الذي المنتجات يمكن أن تضفي مقاومة للمضادات الحيوية أو المطهرات 8. الفئة 1 integrons هي integrons تعافى الأكثر شيوعا من العزلات السريرية 5، حيث اكتسبت مجتمعة أكثر من 130 مختلفة أشرطة المقاومة للمضادات الحيوية الجينات 9. انتشار الطبقة 1 integrons إلى المتعايشة والمسببة للأمراض البكتيريا المرتبطة الإنسان يولد تيارات النفايات البشرية التي تحتوي على أعداد كبيرة من هذه العناصر الجينية 10. يتم الافراج يقدر ب 10 19 البكتيريا التي تحتوي على الطبقة 1 integrons عن طريق مياه الصرف الصحي الحمأة كل ط العامن المملكة المتحدة (11). ولذلك ليس من المستغرب أن الطبقة 1 integrons يمنح المقاومة للمضادات الحيوية يجري حاليا الكشف عن الجراثيم في الطيور البرية والأسماك، وغيرها من 12-14 الحياة البرية الوطنية. الإفراج عن integrons مرة أخرى إلى البيئة يشكل خطرا كبيرا على الصحة العامة، منذ اقتناء أشرطة الجينات الجديدة وإعادة ترتيب معقدة مع عناصر المحمولة الأخرى مازالت تحدث، وخاصة في محطات معالجة مياه الصرف الصحي وغيرها من المسطحات المائية 15-18. البيئة الطبيعية ثم يصبح أرضا خصبة لتجنيد محددات مقاومة جديدة ومسببات الأمراض الانتهازية 19،20. يمكن للبكتيريا التي تحتوي على integron جديدة وسائط جديدة دائرة حول العودة الى المجتمع البشري من خلال المياه والأغذية الملوثة 21،22. مراقبة وسائط البيئية هي استراتيجية رئيسية لفهم وإدارة المقاومة للمضادات الحيوية في 23 في المستقبل. على وجه الخصوص، ينبغي إيلاء الانتباه إلى المواد الغذائية التي تؤكل نيئة أوالمطبوخة قليلا، لأن هذه تمثل أكبر تهديد لنقل عناصر متنقلة جديدة ومسببات الأمراض. في هذا البروتوكول، واتباع نهج مبسط للكشف عن، تحديد وتوصيف الدرجة 1 integrons وأشرطة الجينات المرتبطة بها في المواد الغذائية وترد (الشكل 2). باستخدام مزيج من زراعة والبلمرة المتسلسل (PCR)، integrons يمكن الكشف عنها بسرعة في المجتمعات البكتيرية معقدة والعزلات الفردية. وترد أساليب لتحديد الأنواع من البكتيريا والتشكل والهوية من أشرطة الجينات المرتبطة integron. هذه الطريقة مناسبة لمجموعة واسعة من الأطعمة النباتية والحيوانية، ويتم إعطاء أمثلة على سير العمل النموذجية لكل من أنواع الطعام هذه.

<table><tbody><tr><td>GoTaq Colourless Mastermix</td><td>Promega</td><td>M7132</td><td>Used in all PCRs</td></tr><tr><td>RNAse (Ribonuclease A from bovine pancreas)</td><td>Sigma</td><td>R6513-10MG</td><td>Used in all PCRs</td></tr><tr><td>HinFI restriction enzyme</td><td>Promega</td><td>R6201</td><td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td></tr><tr><td>100 bp ladder</td><td>GE Healthcare</td><td>27400701</td><td>Used as a size standard on all agarose gels</td></tr><tr><td>GelRed DNA stain</td><td>Biotium</td><td>41003</td><td>CAUTION: Personal protection must be worn when handling this material</td></tr><tr><td>Guanidinium thiocyanate</td><td>Life Technologies</td><td>AM9422</td><td>CAUTION: Personal protection must be worn when handling this material</td></tr><tr><td>CLS-TC Solution</td><td>MP Biomedicals</td><td>6540409</td><td>Resuspension solution used at the begining of the genomic DNA extraction</td></tr><tr><td>Lysing Matrix E FastPrep tubes</td><td>MP Biomedicals</td><td>116914500</td><td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td></tr><tr><td>Binding matrix</td><td>MP Biomedicals</td><td>116540408</td><td>Diluted 1:5 with 6 M guanidinium thiocyanate and used in the genomic DNA extraction method.</td></tr><tr><td>Fast Prep machine</td><td>MP Biomedicals</td><td>Number of options available</td><td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit &lt;a href=&quot;http://www.mpbio.com&quot; target=&quot;_blank&quot;&gt;http://www.mpbio.com&lt;/a&gt; for more information</td></tr></tbody></table>

screening foodstuffs for class 1 integrons and gene cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

<html> فحص الثقافات المختلطة والعزلات البكتيرية للintI1 مجموعة التمهيدي HS463a / HS464 PCR يمكن استخدامها للكشف عن وجود للintegron integrase الجينات الدرجة 1، intI1 (الشكل 1). هذه المجموعة التمهيدي يعمل بشكل جيد للكشف عن intI1 في الثقافات ا...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

فحص المواد الغذائية للفئة 1 Integrons وجين كاسيتات

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

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9.0K Views.  Macquarie University. This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs. 

Video: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

موقع محدد الهندسة الكروموسوم البكتيري: ΦC31 كاسيت الموفقة تبادل Integrase (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Bioengineering

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

إنتاج الجينات الحذف في الإشريكيّة القولونية بتوصيل P1 مع أشرطة الكاسيت المهربة مقاومة المضادات الحيوية

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Genetics

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

إثراء والكشف عن الأنماط التوكستريديوم بيرفرينجنز في عينات أغذيه التجزئة

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Immunology and Infection

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

التكامل بوساطة φC31 الموجهة بالموقع وتبادل الكاسيت في نواقل الأنوفيليس للملاريا

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

فحص وعزل بكتيريا C-glycoside-clasinal المعوية

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

Biochemistry

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

منصة الفائق لفحص السالمونيلا spp./دوسنتاريا spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

باستخدام Coculture لكشف كيميائيا وساطة بين الأنواع التفاعلات

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Biology

التعبير الجيني أحادي النسخة لتوصيف أنظمة تدفق الأدوية المتعددة

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

تسليط الضوء على المؤلف: تطوير أبحاث مقاومة المضادات الحيوية باستخدام سلالة بكتيرية تعاني من نقص التدفق ونظام التعبير الجيني أحادي النسخة

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

توصيف أنظمة تدفق الأدوية المتعددة في الراكدة البوماني باستخدام سلالة بكتيرية تعاني من نقص التدفق ونظام التعبير الجيني أحادي النسخة

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

فحص المواد الغذائية للفئة 1 Integrons وجين كاسيتات

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موقع محدد الهندسة الكروموسوم البكتيري: ΦC31 كاسيت الموفقة تبادل Integrase (IMCE)

إنتاج الجينات الحذف في الإشريكيّة القولونية بتوصيل P1 مع أشرطة الكاسيت المهربة مقاومة المضادات الحيوية

إثراء والكشف عن الأنماط التوكستريديوم بيرفرينجنز في عينات أغذيه التجزئة

?????? PCR ????? ??????? ?? ?????????? ????? ???????? ????? ?? ????? ??? ?????? ?? ????????? ??????

التكامل بوساطة φC31 الموجهة بالموقع وتبادل الكاسيت في نواقل الأنوفيليس للملاريا

فحص وعزل بكتيريا C-glycoside-clasinal المعوية

منصة الفائق لفحص السالمونيلا spp./دوسنتاريا spp.

باستخدام Coculture لكشف كيميائيا وساطة بين الأنواع التفاعلات

توصيف أنظمة تدفق الأدوية المتعددة في الراكدة البوماني باستخدام سلالة بكتيرية تعاني من نقص التدفق ونظام التعبير الجيني أحادي النسخة

توصيف أنظمة تدفق الأدوية المتعددة في الراكدة البوماني باستخدام سلالة بكتيرية تعاني من نقص التدفق ونظام التعبير الجيني أحادي النسخة