1. أ.د. عالية عبد الباقي شعيب المملكة العربية السعودية جامعة الملك سعود كلية العلوم قسم البنات والأحياء الدقيقة Mic 623 Plasmids and Antibiotics in Pathogenic Bacteria

2. A Focus on Listeria monocytogenes Listeria monocytogenes (Lm) is the causative bacteria of listeriosis, a very dangerous and often deadly disease. The CDC reports approximately 2,500 cases a year, 500 of which are fatal. This is more than Salmonella and Botulism. بِسْمِ اللهِ الرَّحْمنِ الرَّحِيمِ

3. Due to the ubiquitous nature of the bacteria, it is among the most highly researched and investigated foodborne pathogens in the United States, Canada, and many other countries. Federal and state governments, universities, and industries are actively conducting research.

4. Effectively controlling Listeria monocytogenes is challenging and requires intensive management and resources. Although, the risk of developing listeriosis is low, the consequences are devastating for both consumers and processors. Research over the past decade has concentrated on testing and detection methods, control methods, and risk assessments.

5.  In 1998, one of the largest outbreaks of Listeria monocytogenes occurred with a large hot dog manufacturer.  The result was 15 adult deaths, six stillbirths, and over one million pounds of product recalled.

6. Listeria innocua transformed with an antibiotic resistance plasmid as a thermal- resistance indicator for Listeria monocytogenes ( Document ) WVuJjMzPWRvY3VtZW50cyYzNz1pbmZv. Journal of Food Protection 54(7) 519-523. Listeria innocua PFEI is a chloramphenicol- and erythromycin-resistant organism obtained by electrotransforming L. innocua ATCC 33091 with the plasmid pGK12.

7. L. innocua ATCC 33091 and L. innocua PFEI were more heat resistant between 56 and 66 degC than Listeria monocytogenes F5069 and Scott A when evaluated in sterile phosphate buffer or milk.

8. Decimal reduction times of each L. innocua strain were 1.5 to 3 times longer in either heating menstruum than were D-values of the most heat resistant L. monocytogenes strain studied (F5069). L. innocua PFEI retained the plasmid during heating so that, of 300 survivors evaluated, 100% were resistant to chloramphenicol and 98% were resistant to erythromycin. Thus L. innocua ATCC 33091 or PFEI would be useful indicator organisms to evaluate the lethality of thermal processes with repect to L. monocytogenes.

9. L. innocua PFEI has the advantages that it could easily be selected and enumerated among a large, complex, background microflora, but it may not be appropriate for application in a food processing environemnt. LISTMO http://www.onefish.org/servlet/CDSServlet?status=ND 0zODE5Mi50b3JsaWJJUEUyOTIyMSY2P

10. Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA. Wuenscher MD, Kohler S, Goebel W, Chakraborty T. Institut fur Genetik und Mikrobiologie, Universitat Wurzburg, Federal Republic of Germany. Wuenscher MDKohler SGoebel W Chakraborty T Mol Gen Genet. 1991 Aug;228(1-2):177-82.

11. A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts).

12. An internal fragment of the listeriolysin gene (lisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 x 10(-3) via lisA homology and these cells could be selected at 42 degrees C using a plasmid-encoded erythromycin resistance.

13. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species. PMID: 1909419 [PubMed - indexed for MEDLINE] http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?c md=Retrieve&db=PubMed&list_uids=1909419& dopt=Abstract

14. Institution: KING FAISAL SPECIALIST HOSP Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from Listeria monocytogenes BM4293 among Gram-Positive and Gram-Negative Bacteria Emmanuelle Charpentier, Guy Gerbaud, and Patrice Courvalin * Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France Received 19 October 1998/Accepted 3 April 1999 http://jb.asm.org/cgi/content/abstract/181/11/3368

15. We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus.

16. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable.

17. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization.

18. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated. * Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45 68 83 20. Fax: (33) (1) 45 68 83 19. E- mail: pcourval@pasteur.fr.pcourval@pasteur.fr Present address: New York University Medical Center, Department of Cell Biology, New York, NY 10016

19. Subtyping of Listeria monocytogenes on the basis of plasmid profiles and arsenic and cadmium susceptibility J. McLauchlin, M.D. Hampton, S. Shah, E.J. Threlfall, A.A. Wieneke & G.D.W. Curtis The susceptibilities to arsenic and cadmium together with the detection of plasmid DNA were evaluated for use as epidemiological markers for the subtyping of Listeria monocytogenes. Plasmid DNA was detected in 34% of 322 apparently unrelated isolates of L. monocytogenes.

20. The resistance to cadmium and arsenic differentiated 565 apparently unrelated cultures into four groups, the smallest being 5% of cultures resistant to both agents, and the largest (53%) being sensitive to cadmium and resistant to arsenic. The resistance patterns to these agents and the presence of plasmid DNA varied markedly between the serotypes of the cultures. The detection of plasmid DNA was strongly associated with cadmium resistance in serogroup 1/2 cultures, but not within those of serogroup 4.

21. Arsenic resistance was not associated with plasmid DNA. All methods were sufficiently stable to be useful for epidemiological investigations. The techniques described here offer simple methods which can be easily utilized in laboratories without a specialized expertise for this bacterium. http://www.blackwell- synergy.com/doi/abs/10.1046/j.1365- 2672.1997.00238.x

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