Vous êtes sur la page 1sur 13

Genomic stability of Salmonella Typhimurium DT41 kept at different physical conditions

Abstract
Text outline

Ina Lucilia Lindblom (V9351)

By
Ina Lucilia Lindblom, Stud. Med. Vet., Faculty of Life Science, University of Copenhagen, Blowsvej 17, DK1870 Frederiksberg C, Denmark.

Thanks to
Himel Barua, DVM, PhD., Poultry Diseases, Department of Microbiology, Faculty of Life Science, University of Copenhagen, Blowsvej 17, DK-1870 Frederiksberg C, Denmark. Gitte Petersen, Laboratory Assistant, Department of Microbiology, Faculty of Life Science, University of Copenhagen, Blowsvej 17, DK-1870 Frederiksberg C, Denmark.

Background and problem statement


Defining the source and chain of transmission within a community, is essential to control and to plan preventive measures of an outbreak of any bacteria. In Denmark there has been great success in controlling Salmonella outbreaks between 1988 and 2000, and that has led to a low incidence of salmonellosis [4]. However within the past 10 years 19 outbreaks of Salmonella has occurred in broiler breeder flocks. Of these, 13 tested positive for Salmonella Typhimurium DT 41 (SeT DT 41). The high frequency of SeT DT 41, in the flocks, is currently unexplained. MLVA typing done by Litrup et al. discovered great genetic diversity within the SeT DT 41 strains sampled and this led to the hypothesis of two distinct possibilities: Independent introduction at farm level by e.g. wild birds or transmission and persistence of genetically unstable clones. Salmonella Typhimurium DT 41 is known to be prevalent in wild birds [1],[5], but has also been isolated at free-range pig farms [6] and from humans [3]. A study done in Great Britain by Hughes et al. (2009), of different serovars of Salmonella enterica in wild birds shows DT 41 as being predominant in gulls, with sporadic occurrences in other wildfowl [1]. Hughes et al. did an extensive genetic testing of DT 41, DT 56 and DT 40, and found DT 41 lacks the gene sopE associated with enteritis and epidemics in humans. Interestingly it was the only phage type containing the virulence gene pefA [2]. The two isolates of DT 41 had a genetic similarity of 77%, suggesting some genetic mutation in the host, or possibly two different clones [2]. In Brazil SeT DT 41 was the most common Salmonella typhimurium phage type found by Tavechio et al. 2009 with a prevalence of 22,6%. These samples originated mostly from humans but also from non-human sources such as animals, environment and food. Again there was diversity among all strains with a similarity of approximately 70% [3]. Majtan et al. 2005 isolated 5,8% SeT DT 41 of 154 SeT samples from humans , between 2003 and 2004. This study concentrates on the genetic stability of SeT DT 41 at different physiological condition in vitro and in an in vivo environment. We wanted to see if some mutations or alterations occur at a genetic level, using plasmid extraction and pulsed-field gel electrophoresis, to explain the re-occurrence of SeT DT 41 in broiler breeder flocks.

Index

Introduction
Milj Bedding material, dust, feed and the birds themselves are all potential carriers of Salmonella. Top-down eradication and extensive testing programs along with slaughter of any infected bird is part of the national program to control Salmonella in broiler breeder flocks. To ensure no contamination survives between flocks, all organic material is removed, the poultry houses cleaned and disinfected and finally left on a resting period of 10-14 days [4]. In spite of this SeT DT 41 has persisted and infected birds are still an issue. We set on testing dust and feed pellet samples along with the in vivo, to best imitate the actual environment. Teori plasmid A plasmid is an extrachromosomal circular DNA molecule within the bacteria, with at least one origin of replication. When bacteria divide, the plasmids within the bacteria are copied and passed on to the daughter cells. Strains from the same clonal line should therefore contain the same plasmids [7]. This theory is the basis for plasmid profiling, where two or more strains can be compared on the basis of their plasmids. However this is not always the case, since plasmids can be transferred via conjugation and some plasmids do not transfer at all. As a result some strains can contain different plasmids or different clonal lines can contain the same plasmids. In general plasmid profiling is considered valid in epidemic strains [7], but this is why we have chosen to submit our strains to PFGE in addition to plasmid profiling. Teori pulsed field

Methods and Materials


Four strains were selected whereas one was from human origin and three from poultry (table 1). MSRV (Modified semisolid Rappaport Vassiliadis), Brilliant green agar, Blood agar were used for isolation of bacteria. After growing bacteria on blood agar, a single colony of salmonella was transferred to LB media and incubated overnight at 37C. For preservation, 700l of overnight culture was mixed with 300l of 50% glycerol in a sterile preservation vial and kept at -80C.

Dust and Pellet The dust and pellet samples were divided in groups (n=19) placed at 5, 20 and 41C respectively. Some pellet samples survived throughout the experiment lasting 25 weeks, while the dust samples failed to be isolated after week 3 out of the 6 weeks isolation was done (table 2). For details see appendix 2.
Strain ID s and sample overview are outlined in table 1 (data provided by Himel Barua).

In vivo Birds of three different age groups 30, 40 and 60 weeks-old were placed separately in three different boxes. Each age group consisted of 11 birds. Two isolates of DT41 were used, one from poultry origin while the other one was a human isolate. The challenge inoculum was prepared from the overnight broth culture and all the birds were inoculated orally with a concentration of 108 CFU of bacteria per ml. To assess faecal

excretion of DT41, cloacal swabs were collected everyday for the first week from each bird and then weekly intervals. The birds were observed for 3 weeks from the day of primary infection. At the termination of the experiment, the birds were sacrificed and subjected to post-mortem examination to observe any gross pathological changes. Caecal tonsils from each bird were collected. Both cloacal swabs and caecal tonsils were checked for Salmonella following classical bacteriological procedures. Finally the positive isolates were preserved at 80C using 50% glycerol.

Plasmid extraction
Plasmid extraction was carried out according to Kado & Liu (1981) on 42 isolates. One ml of overnight shaken culture in LB-broth incubated at 37, was used and separated in 0,8% Seakem LE agarose. The gels were stained 15 minutes in ethidium-bromide and photographed using UV light.

Pulsed-field gel electrophoresis


Pulsed-field gel electrophoresis (PFGE) was done as described by Her skal tekst om Pulsed-field ind

Results
Ved plasmid profilering s du x antal profiler se Figur x for detaljer. For Pulsed-field se Figur y for detaljer. MLVA typing result provided by Himel showed .

Discussion
Detailed description of the problems encountered during the study Beskrivelse af jeres forudstninger og fund under forsgene.
Her lgges ogs interessante billeder ind med overvejelser om, hvorfor de ser sdan ud.

(Forslag: Evaluation of the results) (Forslag: Hvad kan man bruge resultatet/observationerne til?) Undersgelse af om disse bakterier er potentielt human patogene. Det violente gen der giver sygdomme hos mennesker ikke er fundet i den engelske undersgelse. Mske er risikoen strre ved smitte fra vilde fugle.

Reference list
(1) Pennycott TW, Park A, Mather HA. Isolation of different serovars of Salmonella enterica from wild birds in Great Britain between 1995 and 2003. Vet Rec. 2006 Jun 17;158(24):817-20. (2) LA Hughes, S Shopland, P Wigley, Hannah Bradon, A Howard Leatherbarrow, Nicola J Williams, Malcolm Bennett, Elizabeth de Pinna, Becki Lawson, Andrew A Cunningham and Julian Chantrey. Characterisation of Salmonella enterica serotype Typhimurium isolates from wild birds in northern England from 2005 2006. Biomedcentral.com/1746-6148/4/4 - BMC Veterinary Research, 2008. (3) Tavechio AT, Fernandes SA, Ghilardi AC, Soule G, Ahmed R, Melles CE. Tracing lineage by phenotypic and genotypic markers in Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- and Salmonella Typhimurium isolated in state of So Paulo, Brazil. Mem Inst Oswaldo Cruz. 2009 Nov;104(7):1042-6. Laboratrio de Bacteriologia, Enteropatgenos do Instituto Adolfo Lutz, So Paulo, SP, Brasil. atavechio@ial.sp.gov.br (4) Henrik C. Wegener,* Tine Hald,* Lo Fo Wong,* Mogens Madsen,* Helle Korsgaard,* Flemming Bager,* Peter Gerner-Smidt, and Kre Mlbak , *)Danish Veterinary Institute, Copenhagen, Denmark; and Statens Serum Institut, Copenhagen, Denmark. Salmonella Control Programs in Denmark. Emerging Infectious Diseases, Volume 9, Number 7, July 2003. (5) Palmgren H, Aspn A, Broman T, Bengtsson K, Blomquist L, Bergstrm S, Sellin M, Wollin R, Olsen B. Department of Infectious Diseases, Ume University, Ume, Sweden. Salmonella in Black-headed gulls ( Larus ridibundus); prevalence, genotypes and influence on Salmonella epidemiology. Epidemiol. Infect. (2006). 134, 635-644 doi: 10.1017/S0950268805005261 . (6) Annette Nygaard Jensen,1* Anders Dalsgaard,2 Anders Stockmarr,1 Eva Mller Nielsen,3, and Dorte Lau Baggesen1. Danish Institute for Food and Veterinary Research, Blowsvej 27, DK-1790 Copenhagen V,1 The Royal Veterinary and Agricultural University, Blowsvej 17, DK-1870 Frederiksberg C,2 Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark3. Survival and Transmission of Salmonella enterica Serovar Typhimurium in an Outdoor Organic Pig Farming Environment. Applied and Environmental Microbiology, March 2006, p. 1833-1842, Vol. 72, No. 3 0099-2240/06/$08.00+0 doi:10.1128/AEM.72.3.1833-1842.2006. (7) J.E. Olsen, D.J. Brown, M.N. Skov and J.P. Christensen, Department of veterinary Microbiology, The Royal Veterinary and Agricultural University, Blowsvej 13, DK-1870 Frederiksberg C, Denmark. Bacterial typing methods suitable for Epidemiological Analysis. Applications in Investigations of Salmonellosis among Livestock. Veterinary Quarterly December 1993: volume 15 no. 4: 125-35 (8) Barbara Joppa, Samantha Li, Scott Cole, Sean Gallagher. HIS Laboratories, Hoefer Scientific Instruments, San Francisco, CA. Pulsed Field Electrophoresis for Separation of Large DNA. Published in Probe Volume 2(3): Fall 1992. (9) C. I. Kado* and S.-T. Liu. Department of Plant Pathology, University of California, Davis, California 95616. Rapid Procedure for Detection and Isolation of Large and Small Plasmids. JOURNAL OF BACTERIOLOGY, Mar. 1981, p. 1365-1373 Vol. 145, No. 3

Appendix 1
Pulsed-field Gel electrophoreses (PFGE)
Protocol for Bacteria Salmonella Typhimurium During the whole process: Use gloves Day 1 Inoculate bacteria in large test tubes containing 10 ml LB broth, prepared the day before to make sure it is sterile. Incubate overnight at 37o in the incubator. If there are problems getting a high enough concentration of bacteria, incubation with a shaking can be advantageous. Day 2 Remove the test tubes from the incubator. Vortex the test tubes before transferring to 15 ml centrifuge tubes, each marked with sample numbers.
Each tube is centrifuged in a cooling centrifuge (10 degrees Celsius) for 10 minutes at 4000 rpm. Remove the supernatant and add 5 ml of CSB before vortexing to resuspend the pellet. (Make sure the OD machine is turned on at least 15 minutes before use). Centrifuge again in a cooling centrifuge (degrees Celsius) for 10 minutes at 4000 rpm. Remove the supernatant and add 2 ml CSB before vortexing to resuspend the pellet.

Preparing agarose While the samples are being centrifuged prepare the agarose for the blocks.
20 ml agarose recipe for blocks: 0,20 g 1% Seakem Gold agarose + 2 ml 10% SDS + 18 ml TE buffer. Heat the agarose solution in a microwave 3*10 seconds until the solution is homogenous and tranperent. Place the agarose in a water bath at 55-56o until use. If there is any left it can be reheated for repeated use.

Maesuring OD Set the OD machine at 610 nm.


Use 1 ml of blank CSB to zero-base the OD machine. Maesure OD on 1 ml of each sample. OD should be between 0.9 and 1.0, if the OD is higher or lower calculate the amount of ONB (overnight broth) to either dilute or strengthen the concentration of the ONB to get 400 l sample with the correct amount of bacterial DNA. See example below. If OD is higher the 1: (1,5 is used in this example)

 

Because the OD was higher we dilute it with 400 - 267 = 133 l CSB, this will give the desired end amount of 400 l with the correct OD. If the OD is lower centrifuge again and remove the required amount of CSB to reach the concentration of OD=1.

Preparing the plugs 400 l sample is transferred to an Eppendorf tube marked with corresponding sample number.
20 l proteinase K (20 mg/ml) is added to each Eppendorf tube. Prepare plug molds. Use one plug mold for 2 samples. Each plug mold is taped in the bottom and marked with sample numbers. The plug molds can either be secured on a glass slide or a platic tray. <billede> While the agarose is still in the water bath, pipette 400 l agarose aolution into the Eppendorf tube containing sample and mix 2 or 3 times before transferring the mixture to the plug molds. Do 4 plugs for each sample. When transferring into the plug molds, be sure to have a steady hand and transfer very slowly to avoid bubbles. Let the plugs cool off in the molds for 15-20 minutes at room temperature or at 4oC.

Buffer master mix Mark new 15 ml centrifuge tubes for each sample.
Recipe for buffer master mix for 1 sample: 5 ml cell lysis buffer + 25 l proteinase K (20mg/ml). The master mix is prepared in a 50 ml centrifuge tube with foot, and 5 ml is transferred to each 15 ml centrifuge tube.

Preparing and transferring the blocks Use the scalpel to remove excess agarose from the top of the mold. Use 70% ethanol on the scalpel between each sample to avoid contamination of the samples.
With a spatula, carefully push the blocks into the 15 ml centrifuge tubes containing the master mix. Make sure all the blocks are submerged. Incubate the blocks in a water bath at 55-56o with a shaking of 70 - 90 rpm for a minimum of 2 hours or alternatively overnight.

Washing All the samples need to be washed 2 times in dH2O and 4 times in TE buffer, with an incubation time of 10 minutes between each washing in a water bath 55-56o 70 - 90 rpm.
Warm 10 ml dH2O /sample and 40 ml TE buffer/sample in a water bath at 55-56o.

I found the best washing technique was to use a spatula and hold it with your index finger to avoid loss of blocks and remove fluid as fast as possible. You can further reduce loss of blocks be passing the removed fluid through a laboratory film with a small hole in the middle. Alternatively a pipette a pipette or a scalpel can be used to remove excess fluid. <Billede> After incubation in water bath, the buffer master mix is removed and 5 ml of dH2O is added to the tubes. Incubate in water bath for 10 minutes. Remove dH2O and add 5 ml of new dH2O. Incubate in bath for 10 minutes. Remove dH2O and add 5 ml of TE buffer. Incubate in bath for 10 minutes. Remove TE buffer and add 5 ml of new TE buffer. Incubate in bath for 10 minutes. Remove TE buffer and add 5 ml of new TE buffer. Incubate in bath for 10 minutes. Remove TE buffer and add 5 ml of new TE buffer. Incubate in bath for 10 minutes. Remove the last TE buffer and add 4 ml of TE buffer. The blocks can be held at 4o until use, and need not be digested immediately.

Day 3 Digesting Mark preservation tube for each sample and for Salmonella Brnderup, if used as marker.
One block from each 15 ml centrifuge tube is carefully removed with the bended end of a spatula and placed on a glass slide or a sterile Petri dish with graph paper underneath. Remove the excess fluid with some blotting paper. With a sharp scalpel cut the block into pieces of 2 - 2.5 mm in size, depending on how much DNA you wish to have in your gel. The most important thing is that all plugs are the same size, when placed on the comb. The plug pieces are then transferred to the preservation tubes containing buffer mix. We use XBA1 (20U/l)to digest our samples and the following recipe to make the buffer mix: Milli Q l/plug slice: 180 l

NE4+BSA l/plug slice: Total:

20 l 200 l/tube

The plug slices are then incubated at room temperature for 15 minutes with buffer mix. Carefully remove the buffer mix with a pipette while tilting the preservation tube, to remove the fluid without disturbing the plug slices. Add enzyme buffer using enzyme buffer recipe: Milli Q l/plug slice: NE4+BSA l/plug slice: XBA1 l/plug slice: Total: 177.5 l 20 l 2.5 l 200 l/tube

Incubate the preservation tubes containing enzyme mix at 37oC for 2 hours.

Preparing the gel Small gel:


Big gel:

100 ml (1 g% Seakem Gold agarose in 100 ml 0.5xTB buffer) 150 ml (1,5 g% Seakem Gold agarose in 150 ml 0.5xTB buffer)

Melt the gel 2.5 x 1 minute in the microwave until the gel is homogeneous and transparent. coll of in water bath at 55-56oC. Preparing the TB buffer for PFGE machine. 100 ml 10xTB up to 2 liters dH2O Assemble the sled and comb. <billide> After incubation one plug slice from each preservation tube is selected and put on a tooth of the comb. Keep the plug lices moist with TE buffer. If the plug slices dry they reduce in size and the DNA can potentially be damaged. leave space on each end of the comb for the Low Range PFGE marker. <billede> If Samonella Brnderup is used, it should be placed next to the Low Range PFGE marker, and if running a big gel, it can be advantageous to place one in the middle of the comb as well. When all plugs are placed on the comb, a drop of agarose is used to fasten the plugs to the comb (see pictures above). Let the agarose plugs dry for 5 minutes. Put the comb in place in the sled and pour in the agarose slowly to avoid bubbles, save a bit to fill up the wells after after the comb is removed.

Let the gel harden for 20-30 minutes. Remove the comb gently and close the wells with the last agarose using a pipette. Let it dry for 5 minutes. Remove the black slide from the sled and use a napkin to dry off any agarose under the slide. The gel is now ready to be put in the PFGE machine. <billed>

Proram Block 1
Initial switch time 2.2 seconds End switch 54.4 seconds Run time 19 hours Volts 6 Included angle 120 Buffer: 2 liters 0.5xTB <billed>

Day 4 Picture Remove gel from the PFGE machine and carefully push it off the black slide onto a plastic tray and transfer it to 1% ethidiumbromid.
After 30 minutes transfer the gel to the tub containing water placed next to the ethidiumbromid and let it stay for 60 minutes. The gel can now be put in the machine to take a picture. Use the guide next to the computer.

Recipes 100 ml 0.5xTB: 5 ml 10xTB and fill up with autoclaved distilled water to 100 ml.
150 ml 0.5xTB: 7.5 ml 10xTB and fill up with autoclaved distilled water to 150 ml. 2 liters 0.5xTB: 100 ml 10xTB and fill up with autoclaved distilled water to 2000 ml. 5 ml Proteinase K (20mg/ml): Weigh of 0.1g proteinase K and add 5 ml distilled water. 500 l added to 10 Eppendorf tubes and kept in the freezer. NE4+BSA: For 1.5 ml NE4 add 150 ml BSA and keep in the freezer.

Preparations Day 1 (to be used day 2) CSB + cell lysis buffer + TE buffer + 3 liters of autoclaved dH2O. Day 2(to be used day 3) 10xTB buffer + Milli Q autoclaved + 1 liter autoclaved dH2O to clean the machine
Enzyme, BSA and NE4 are bought. Proteinase K will have to be prepared the same day as used.

Vous aimerez peut-être aussi