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Characterisation of Food Associated Bacteria by DNA based Methods, with Special Reference to Enterobacteriaceae

Olsson, Crister LU (2003)
Abstract (Swedish)
Popular Abstract in Swedish

Vissa E. coli bakterier kan vara utrustad med arvsanlag (gener) som kodar för vidhäftningsfaktorer, som gör att den kan binda till tarmslemhinnan, eller toxiner som kan orsaka olika typer av tarmsjukdom som diarré. En annan faktorer som kan bidra till att en bakterie kan orsaka infektion är genkassetter, som kodar för en rad av olika gener, som t.ex för upptag av järn, nödvändigt för organismens metabolism. Närvaro av sådana gener undersöktes i livsmedel med PCR teknik, dvs generna mångfaldigas så att de blir möjliga att detektera med olika typer av infärgningsmetoder. Bakterier nära besläktade med E. coli studerades också med olika genetiska eller s.k. DNA baserade metoder, där man utnyttjar... (More)
Popular Abstract in Swedish

Vissa E. coli bakterier kan vara utrustad med arvsanlag (gener) som kodar för vidhäftningsfaktorer, som gör att den kan binda till tarmslemhinnan, eller toxiner som kan orsaka olika typer av tarmsjukdom som diarré. En annan faktorer som kan bidra till att en bakterie kan orsaka infektion är genkassetter, som kodar för en rad av olika gener, som t.ex för upptag av järn, nödvändigt för organismens metabolism. Närvaro av sådana gener undersöktes i livsmedel med PCR teknik, dvs generna mångfaldigas så att de blir möjliga att detektera med olika typer av infärgningsmetoder. Bakterier nära besläktade med E. coli studerades också med olika genetiska eller s.k. DNA baserade metoder, där man utnyttjar skillnader i nukleinsyra sammansättningen i själva DNA strängen. Avsikten var att få fram kompletterande metoder för en snabbare och säkrare identifiering av den bakterieflora som finns i livsmedel. Dessutom studerades sammansättningen av bakteriefloran i fläskkött med en sådana här metoder. Bakteriefloran i kött har blivit väl studerad i tidigare undersökningar. Man har då man använt sig av odlingsmetoder, dvs. man odlar fram och isolerar mikroorganismerna av intresse som sedan identifieras med biokemiska metoder. Nu kan kan man preparera fram bakteriernas DNA från provet istället och med DNA baserade metoder identifiera vilka organismer som finns i provet från början. Fördelen med dessa nya metoder är att man kan påvisa förekomst av bakterier som är svåra att odla i laboratoriet.



Några gener som kodade för toxin eller vidhäftningsfaktorer kunde inte detekteras i de undersökta livsmedlen, som huvudsakligen bestod av nöt- och fläskkött. Däremot kunde vi påvisa förekomsten av en genkassett som kodar för upptag av järn i en till E. coli besläktad bakterie, Serratia liquefaciens. Denna bakterie kan ibland växa till höga antal i proteinrika kylförvarade livsmedel. Detta visar att faktorer, som hos närbesläktade bakteriearter kan bidra till en infektion, kan finnas i mikroorganismer som är vanligt förkommande i kött.



Med hjälp av DNA baserade metoder var det möjligt att identifiera bakterierarter av släkten som Klebsiella, Hafnia, Serratia och Rahnella. Dessa typer av organismer kan bidra till förskämmningen av livsmedel och kan ibland orsaka sjukdom hos personer med ett försvagat immunförsvar. Dessutom var det möjligt att gruppera Rahnella i undergrupper med dessa metoder, vilket kan ha betydelse när man vill spåra en smittkälla för ett livsmedel t.ex.



Analys av fläskkött med DNA baserade metoder visade att bakteriefloran överenstämde ganska väl med vad man tidigare funnit med konventionella odlingsmetoder. Från början är bakteriefloran blandad bestående av både grampositiva och gramnegativa bakterier men övergår under kyllagring till en mer ensartad flora av arter som trivs i kyla som t.ex. Pseudomonas flourescens, Pseudomonas fragi och Pseudomonas lundensis. Analysen visade också förekomst av Eperythrozoon som inte låter sig odlas på vanliga laboratoriemedia. Denna bakterie kan orsaka infektion hos människa, men man har lite kunskap om hur den sprids. (Less)
Abstract
The presence of genes in food, encoding some virulence factors, was studied by PCR, and species of Enterobacteriaceae, associated with food, were studied by the DNA-based methods of TTGE, ribotyping and sequencing. The flora of fresh and chill-stored pork were analysed by a culture-independent approach, using specific amplification of 16S rRNA genes followed by cloning and sequencing.



No evidence for the presence of toxin coding genes related to E. coli was found in meat and fish but a strain of Serratia liquefaciens positive for the Yersinia HPI, was isolated from a sample of beef. Up to now, this is the first Serratia found to be HPI positive but the significance, from a food hygiene point of view, is not clear and S.... (More)
The presence of genes in food, encoding some virulence factors, was studied by PCR, and species of Enterobacteriaceae, associated with food, were studied by the DNA-based methods of TTGE, ribotyping and sequencing. The flora of fresh and chill-stored pork were analysed by a culture-independent approach, using specific amplification of 16S rRNA genes followed by cloning and sequencing.



No evidence for the presence of toxin coding genes related to E. coli was found in meat and fish but a strain of Serratia liquefaciens positive for the Yersinia HPI, was isolated from a sample of beef. Up to now, this is the first Serratia found to be HPI positive but the significance, from a food hygiene point of view, is not clear and S. liquefaciens is only occasionally isolated from clinical specimens.



For the primary purpose of evaluating the TTGE method for identification of Enterobacteriaceae associated with food, strains isolated from meat, fish and milk and human isolates of Klebsiella, a genus that is frequently found in food, were included in the study. TTGE proved to be a tool for differentiating E. coli, E. vulneris, E. cloacae, E. amylovora, H. alvei, K. terrigena, P. agglomerans, R. aquatilis, and distinguished between the clinically important strains of K. pneumoniae and K. oxytoca. In addition, it was possible to identify R. aquatilis genomospecies (GS) 1 and 2 and presumably one additional GS group of this species using TTGE. The closely related species of K. planticola, K. ornithinolytica and E. aerogens were not separated by TTGE. Furthermore, the C. freundii and Y. ruckeri type strains could not be distinguished from each other and S. liquefaciens and S. proteamaculans could not be identified due to diffuse or lack of band on the gel. Ribotyping was useful to subdivide Rahnella aquatilis into genomic subtypes and for confirmation of the TTGE grouping. The strains of H. alvei and S. liquefaciens were genomically defined by ribotyping. Sequencing of 16S rRNA genes was useful for the verification of the identity of Klebsiella isolates and the different Rahnella genomospecies. In addition a protocol was developed for rapid identification of Klebsiella spp. By picking a colony from an agar plate, preparing the DNA for PCR and running TTGE over night, identification was possible within 24 hours.



A culture-independent analysis of the flora of fresh and chill-stored pork gave an overall picture, of the bacterial composition, which was in agreement with cultivation-dependent methods previously used. The initial flora turned out to be diverse in terms of the numbers of genera present, and a change occurred during refrigerated storage into a gram-negative psychrotrophic flora, dominated by Pseudomonas. The method used here also indicated the possibility of detecting bacterial species such as Eperythrozoon, that do not grow on ordinary media. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Associate Professor Ljungh, Åsa, Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Food and drink technology, 16S rRNA, TTGE, ribotyping, Rahnella, Klebsiella, HPI, Enterobacteriaceae, food, Livsmedelsteknik
pages
80 pages
publisher
Division of Food Technology, Lund University
defense location
Sal B, Centre for Chemistry and Chemical Engineering, Getingev. 60, Lund
defense date
2003-10-03 10:30
language
English
LU publication?
yes
id
870a5ac0-e239-4d10-b9c6-5695c3ba482c (old id 466212)
date added to LUP
2007-10-14 14:27:26
date last changed
2016-09-19 08:45:13
@misc{870a5ac0-e239-4d10-b9c6-5695c3ba482c,
  abstract     = {The presence of genes in food, encoding some virulence factors, was studied by PCR, and species of Enterobacteriaceae, associated with food, were studied by the DNA-based methods of TTGE, ribotyping and sequencing. The flora of fresh and chill-stored pork were analysed by a culture-independent approach, using specific amplification of 16S rRNA genes followed by cloning and sequencing.<br/><br>
<br/><br>
No evidence for the presence of toxin coding genes related to E. coli was found in meat and fish but a strain of Serratia liquefaciens positive for the Yersinia HPI, was isolated from a sample of beef. Up to now, this is the first Serratia found to be HPI positive but the significance, from a food hygiene point of view, is not clear and S. liquefaciens is only occasionally isolated from clinical specimens.<br/><br>
<br/><br>
For the primary purpose of evaluating the TTGE method for identification of Enterobacteriaceae associated with food, strains isolated from meat, fish and milk and human isolates of Klebsiella, a genus that is frequently found in food, were included in the study. TTGE proved to be a tool for differentiating E. coli, E. vulneris, E. cloacae, E. amylovora, H. alvei, K. terrigena, P. agglomerans, R. aquatilis, and distinguished between the clinically important strains of K. pneumoniae and K. oxytoca. In addition, it was possible to identify R. aquatilis genomospecies (GS) 1 and 2 and presumably one additional GS group of this species using TTGE. The closely related species of K. planticola, K. ornithinolytica and E. aerogens were not separated by TTGE. Furthermore, the C. freundii and Y. ruckeri type strains could not be distinguished from each other and S. liquefaciens and S. proteamaculans could not be identified due to diffuse or lack of band on the gel. Ribotyping was useful to subdivide Rahnella aquatilis into genomic subtypes and for confirmation of the TTGE grouping. The strains of H. alvei and S. liquefaciens were genomically defined by ribotyping. Sequencing of 16S rRNA genes was useful for the verification of the identity of Klebsiella isolates and the different Rahnella genomospecies. In addition a protocol was developed for rapid identification of Klebsiella spp. By picking a colony from an agar plate, preparing the DNA for PCR and running TTGE over night, identification was possible within 24 hours.<br/><br>
<br/><br>
A culture-independent analysis of the flora of fresh and chill-stored pork gave an overall picture, of the bacterial composition, which was in agreement with cultivation-dependent methods previously used. The initial flora turned out to be diverse in terms of the numbers of genera present, and a change occurred during refrigerated storage into a gram-negative psychrotrophic flora, dominated by Pseudomonas. The method used here also indicated the possibility of detecting bacterial species such as Eperythrozoon, that do not grow on ordinary media.},
  author       = {Olsson, Crister},
  keyword      = {Food and drink technology,16S rRNA,TTGE,ribotyping,Rahnella,Klebsiella,HPI,Enterobacteriaceae,food,Livsmedelsteknik},
  language     = {eng},
  pages        = {80},
  publisher    = {ARRAY(0x93e4138)},
  title        = {Characterisation of Food Associated Bacteria by DNA based Methods, with Special Reference to Enterobacteriaceae},
  year         = {2003},
}