Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level.
BACKGROUND
The emerging threat posed by antibiotic resistance has affected public health systems all over the world. Surveillance of resistant bacteria in clinical settings and identifying them in mixed cultures is of paramount importance and can contribute to the control of their spreading. Culture-independent monitoring approaches are highly desirable, since they yield results much faster than traditional susceptibility testing. However, many rapid molecular methods like PCR only detect the sole presence of a potential resistance gene, do not provide information regarding efficient transcription, expression and functionality and, in addition, cannot assign resistance genes to species level in mixed cultures.


METHODS
By using plasmid-encoded TEM β-lactamase mediated ampicillin resistances as a proof of principle system, we (1) developed a fluorescence in situ hybridization-test (FISH) capable to detect the respective mRNAs, (2) implemented an immunofluorescence test to identify the corresponding proteins and (3) compared these two microscopic tests with an established colorimetric nitrocefin assay to assess the enzymatic activity.


RESULTS
All three methods proved to be suitable for the testing of antibiotic resistance, but only FISH and immunofluorescence were able to differentiate between susceptible and resistant bacteria on the single cell level and can be combined with simultaneous species identification.


CONCLUSIONS
Fluorescence in situ hybridization and immunofluorescence tests are promising techniques in susceptibility testing since they bridge the gap between the slow, but accurate and sound cultural methods and molecular detection methods like PCR with much less functional relevance.
Department of Biological Safety, Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany. alexander.rohde@bfr.bund.de.|Department of Biology, Chemistry and Pharmacy, Free University Berlin, Takustr. 3, 14195, Berlin, Germany. alexander.rohde@bfr.bund.de.
Alexander
A
Rohde
Department of Biological Safety, Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany.
Jens Andre
JA
Hammerl
Department of Biological Safety, Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany.
Sascha
S
Al Dahouk
Antibiotic resistance; Fluorescence in situ hybridization; Plasmids; β-Lactamases
Philos Trans R Soc Lond B Biol Sci. 2015 Jun 5;370(1670):20140080
25918439
Vet Microbiol. 2014 Jul 16;171(3-4):279-83
24674741
Gene. 1981 Jan-Feb;13(1):25-35
6263753
J Clin Microbiol. 2008 Nov;46(11):3842-4
18753354
N Engl J Med. 2009 Jan 29;360(5):439-43
19179312
Rev Sci Tech. 2013 Aug;32(2):383-92
24547644
Clin Microbiol Infect. 2006 May;12(5):410-7
16643516
FEMS Microbiol Lett. 2006 Jan;254(2):217-25
16445748
BMC Microbiol. 2009 Mar 02;9:46
19254378
Int J Antimicrob Agents. 2013 Jun;42 Suppl:S25-8
23684003
Clin Microbiol Infect. 2008 Jun;14(6):614-6
18397333
Gene. 1982 Oct;19(3):259-68
6295879
Biotechniques. 1995 Jul;19(1):130-5
7669286
Food Microbiol. 2015 Apr;46:395-407
25475309
Gut. 2000 May;46(5):608-14
10764702
Nat Protoc. 2013 Jun;8(6):1100-13
23680982
Biomed Res Int. 2014;2014:375681
25343142
Nat Methods. 2008 Oct;5(10):877-9
18806792
Nat Genet. 2011 Jun;43(6):554-60
21532574
FEMS Microbiol Lett. 2003 Nov 21;228(2):187-91
14638423
Antimicrob Agents Chemother. 1990 Nov;34(11):2184-92
2073109
Int J Hyg Environ Health. 2000 Mar;203(1):77-82
10956593
Int J Antimicrob Agents. 2007 Mar;29(3):344-7
17276039