PMID- 19446419
OWN - NLM
STAT- MEDLINE
DCOM- 20090908
LR  - 20191210
IS  - 1618-0984 (Electronic)
IS  - 0723-2020 (Linking)
VI  - 32
IP  - 5
DP  - 2009 Aug
TI  - In situ detection of antibiotic-resistance elements in single Bacillus cereus 
      spores.
PG  - 323-33
LID - 10.1016/j.syapm.2009.03.003 [doi]
AB  - Rapid detection of Bacillus spores is a challenging task in food and defense 
      industries. In situ labeling of spores would be advantageous for detection by 
      automated systems based on single-cell analysis. Determination of 
      antibiotic-resistance genes in bacterial spores using in situ labeling has never 
      been developed. Most of the in situ detection schemes employ techniques such as 
      fluorescence in situ hybridization (FISH) that target the naturally amplified 
      ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a 
      plasmidic or chromosomal origin and is present in low copy numbers in the cell. 
      The main challenge in the development of low-target in situ detection in spores 
      is the permeabilization procedure and the signal amplification required for 
      detection. This study presents permeabilization and in situ signal amplification 
      protocols, using Bacillus cereus spores as a model, in order to detect 
      antibiotic-resistance genes. The permeabilization protocol was designed based on 
      the different layers of the Bacillus spore. Catalyzed reporter deposition 
      (CARD)-FISH and in situ polymerase chain reaction (PCR) were used as signal 
      amplification techniques. B. cereus was transformed with the high copy number 
      pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to 
      chloramphenicol and erythromycin, respectively. In addition, a 
      rifampicin-resistant B. cereus strain, conferred by a single-nucleotide 
      polymorphism (SNP) in the chromosome, was used. Using CARD-FISH, only the high 
      copy number plasmid pC194 was detected. On the other hand, in situ PCR gave 
      positive results in all tested instances. This study demonstrated that it was 
      feasible to detect antibiotic-resistance genes in Bacillus spores using in situ 
      techniques. In addition, in situ PCR has been shown to be more sensitive and more 
      applicable than CARD-FISH in detecting low copy targets.
FAU - Laflamme, Christian
AU  - Laflamme C
AD  - Institut Universitaire de cardiologie et de pneumologie, Centre de recherche, 
      Hopital Laval, Universite Laval, 2725 Chemin Ste-Foy, Ste-Foy, Quebec, Canada G1V 
      4G5.
FAU - Gendron, Louis
AU  - Gendron L
FAU - Turgeon, Nathalie
AU  - Turgeon N
FAU - Filion, Genevieve
AU  - Filion G
FAU - Ho, Jim
AU  - Ho J
FAU - Duchaine, Caroline
AU  - Duchaine C
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20090514
PL  - Germany
TA  - Syst Appl Microbiol
JT  - Systematic and applied microbiology
JID - 8306133
RN  - 0 (DNA, Bacterial)
SB  - IM
MH  - Bacillus cereus/*genetics
MH  - DNA, Bacterial/*genetics
MH  - *Drug Resistance, Bacterial
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Plasmids
MH  - Polymerase Chain Reaction/*methods
MH  - Sensitivity and Specificity
MH  - Spores, Bacterial/*genetics
EDAT- 2009/05/19 09:00
MHDA- 2009/09/09 06:00
CRDT- 2009/05/19 09:00
PHST- 2008/11/19 00:00 [received]
PHST- 2009/03/12 00:00 [revised]
PHST- 2009/03/18 00:00 [accepted]
PHST- 2009/05/19 09:00 [entrez]
PHST- 2009/05/19 09:00 [pubmed]
PHST- 2009/09/09 06:00 [medline]
AID - S0723-2020(09)00046-0 [pii]
AID - 10.1016/j.syapm.2009.03.003 [doi]
PST - ppublish
SO  - Syst Appl Microbiol. 2009 Aug;32(5):323-33. doi: 10.1016/j.syapm.2009.03.003. 
      Epub 2009 May 14.