Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level
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.
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.
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.
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.
KeywordsAntibiotic resistance Fluorescence in situ hybridization β-Lactamases Plasmids
fluorescence in situ hybridization
polymerase chain reaction
reverse transcription PCR
minimal inhibitory concentration
- Y. pseudotuberculosis
- E. coli
- K. pneumoniae
- Y. enterocolitica
American Type Culture Collection
The emergence of antibiotic resistance is threatening the public health in industrialized as well as developing countries, resulting in therapy failure and increased health-care expenditures [1, 2]. Monitoring the occurrence of antibiotic resistance is one important component to control its spreading and, consequently, numerous surveillance programs have been implemented [3, 4, 5, 6]. The susceptibility of bacterial isolates towards certain antimicrobials is mainly assessed by slow cultural methods, which require the availability of pure isolates. However, in clinical settings rapid susceptibility testing is extremely crucial to initiate appropriate therapeutic measures because any delay might increase morbidity, mortality and long-term sequelae; therefore, alternative testing methods are gaining importance in hospital care . Screening ubiquitous biofilms in hospitals consisting of multiple different bacterial species or blood samples containing mixed cultures for resistance genes can be carried out by PCR-testing; however, detected resistance genes can neither be attributed to a specific species, nor can it prove a functional resistance. Therefore, culture-independent assays on the single cell level, preferably coupled with the simultaneous species identification, are highly desirable, not only for academic purposes, but also in clinical or environmental microbiology. β-lactamases are one of the most frequently encountered mediators of antimicrobial resistance. Among them, TEM β-lactamases encoded on plasmids represent especially interesting targets because mobile elements like plasmids can easily spread antibiotic resistance. To detect the presence of these resistance elements, three culture-independent methods appear to be capable of fulfilling this task. Firstly, the detection of TEM β-lactamase mRNA transcripts can be performed by a modified version of fluorescence in situ hybridization (FISH), which was initially developed for eukaryotes and has recently also been successfully used for prokaryotes [8, 9, 10]. Previous attempts using FISH were mainly restricted to detect single mutations in the highly abundant rRNAs conferring resistance to antibiotics targeting ribosomes, e.g. macrolides [11, 12]; however, by applying dozens of probes simultaneously (instead of only one) mRNAs with much lower concentrations can be detected as well. In parallel with mRNA detection, FISH can be used to simultaneously identify the bacterial species based on their ribosomal RNA [7, 11]. Secondly, immunofluorescence stainings of the proteins with specific antibodies represent a further alternative and can also be used for concurrent species identification . Thirdly, chromogenic substrates like nitrocefin offer a way to test the enzymatic activity of TEM β-lactamases [14, 15]. Apart from these methods, reverse transcription PCR assays (RT-PCR) are an additional possibility enabling the detection of efficient transcription [16, 17, 18]. However, in contrast to FISH and immunofluorescence stainings, RT-PCR cannot detect resistance elements on the single cell level. In this work, we established a FISH assay to detect TEM mRNAs encoded on different kind of plasmids and, additionally, an immunofluorescent staining to detect the corresponding proteins and compared these methods with the traditional nitrocefin assay to screen for functional β-lactamases.
Strains and cultivation
Escherichia coli strain DH5α carrying either the high copy number plasmids pLitmus38 (AMPr) and pUC18 (AMPr) or the medium copy number vector pBR328 (AMPr, TETr, CHLr) with a low plasmid stability were used as well as the E. coli reference strain ATCC 35218, a reference strain producing TEM-1 β-lactamases [19, 20, 21]. On the plasmids pLitmus38, pUC18 and pBR328 a TEM-1 gene confers resistance towards ampicillin. Susceptibility of these bacterial strains was tested by Etest® (bioMerieux, France) according to manufacturer specifications and all AMPr E. coli strains possessed MICs >256 µg/ml. As negative controls, DH5α (MIC <1.5 µg/ml) and GeneHogs (MIC <2 µg/ml; Thermo Fisher Scientific, USA) without plasmids were used as susceptible E. coli strains as well as Y. pseudotuberculosis ATCC 29833 (MIC <0.125 µg/ml). Two clinical Klebsiella pneumoniae isolates, K2 with an intermediate resistance (MIC <24 µg/ml) and the highly resistant strain My6107 (MIC >256 µg/ml), and Yersinia enterocolitica DSM 13030 (MIC >256 µg/ml) were used as ampicillin-resistant strains expressing non-TEM β-lactamases. E. coli and K. pneumoniae were grown in LB medium at 37 °C, Y. enterocolitica and Y. pseudotuberculosis at 28 °C. To exhibit antibiotic stress, bacterial cultures were grown in LB medium containing ampicillin in a concentration of 100 µg/ml.
In addition, 25 E. coli isolates from different environmental samples and with different TEM-variants (Additional file 1: Figure S1) were used to verify the inclusivity and sensitivity of the established FISH and immunofluorescence assays. To test whether the newly developed assays are applicable for mixed bacterial cultures, samples containing different species (Salmonella enterica, thermophilic Campylobacter spp., Listeria spp., Y. enterocolitica, E. coli O157) were prepared as described earlier .
Oligonucleotides used to detect TEM mRNA and Enterobacteriaceae (as a positive hybridization control)
Enterobacteriaceae (adapted with minor modifications from Bohnert et al. )
Competitor for Enterobacteriaceae detection (adapted with minor modifications from Bohnert et al. )
TEM β-lactamase mRNA
Bacterial cultures were prepared as described for FISH. After drying on a glass slide, the bacteria were permeabilized with lysozyme (Carl Roth, 10 mg/ml) for 7 min and afterwards rinsed shortly with water. Samples were then blocked with blocking buffer [2 % of bovine serum albumin in PBS (Sigma-Aldrich, USA)] for 1 h at room temperature. Subsequently, the primary antibody [anti-(TEM) β-lactamase ab12251 (mouse); abcam, United Kingdom], diluted 1:200 in blocking buffer, was added and incubated either overnight at 6 °C (for sequential stainings) or 1 h at room temperature. Slides were rinsed shortly with water and washed three times with PBS and, finally, with blocking buffer for 3 min each. Slides were then incubated with the secondary antibody (goat anti-mouse IgG-Alexa Fluor® 488 ab150117, abcam), diluted 1:300 in blocking buffer, for 1 h at room temperature. Slides were again rinsed shortly with water and washed three times with PBS for 3 min each, once more rinsed with water, air-dried and embedded in Roti®-Mount FluorCare DAPI. Each bacterial strain was stained in three independent immunofluorescence assays.
Microscopy was carried out with an AxioScope fluorescence microscope using a 100× N-achroplan Ph3 M27 oil objective (Zeiss, Germany). Images were acquired by the AxioCam MRm and further processed for overlay of different fluorophore channels by using the imaging software ZEN 2012 (Zeiss).
Nitrocefin (Merck Millipore, Germany) was dissolved in DMSO (PanReac Applichem, Germany) in a concentration of 5 mg/ml. 50 µl of this stock solution was added to 950 µl PBS. 50 µl of this nitrocefin working solution was added to 150 µl of a bacterial culture. Alternatively, a colony was picked from an agar plate and suspended in 50 µl nitrocefin working solution on a glass slide. A colour change from yellow to red was considered as proof for functional β-lactamases. Each bacterial strain was tested three times.
Results and discussion
Immunofluorescence staining of the TEM β-lactamase protein showed a more even signal distribution among the bacterial cells than the RNA-signals determined by mRNA FISH (compare Fig. 1a, b). Stationary cultures also showed stronger signals compared to exponentially growing bacteria. As expected for a protein which is secreted in the periplasm, ring-shaped halo-like structures around the cells were observed, especially for pUC18 E. coli strains (Fig. 1b, right), whereas strains harbouring pLitmus38 and pBR328 as well as E. coli ATCC 35218 showed protein accumulations mainly in the cell poles (Fig. 1b, left). In accordance with FISH, antibody staining produced stronger signals for pLitmus38 and pUC18 than for pBR328 and E. coli ATCC 35218. Bacteria without a TEM gene were not stained by immunofluorescence (Additional file 3: Table S1).
To verify that both assays can be applied to environmental samples, 25 E. coli strains with different TEM-variants (TEM-1, TEM-30, TEM-52) were tested. All 25 isolates were detected by immunofluorescence and showed protein accumulations mainly in the cell poles. However, four strains exhibited a rather weak antibody staining. Likewise, the FISH signals of two strains were too weak to be reliably detected (Additional file 1: Figure S1). However, a combination of both methods enabled clear results. This combined testing might be useful to avoid false-negative results and is especially advisable if samples with strong background fluorescence are examined like food matrices or filtrates [22, 24].
The nitrocefin assay is an established general assay to identify the presence of most types of β-lactamase producing strains. All E. coli strains with the high copy number plasmids pUC18 and pLitmus38 as well as K. pneumoniae My6107 were rapidly tested positive for β-lactamase in a few minutes, both by colony smear as well as in liquid cultures, whereas all susceptible strains produced negative results (Additional file 4: Figure S3). Interestingly, the colour change for Y. enterocolitica, K. pneumoniae K2, E. coli ATCC 35218 and strains harbouring the plasmid pBR328 with a low stability and a relatively weak β-lactamase production  was less pronounced, especially in liquid cultures without selection pressure, and took significantly longer than for the resistant E. coli strains with high copy plasmids, which showed a much more rapid substrate turnover (Additional file 4: Figure S3). We also tried to use nitrocefin to visualize β-lactamase activity on the single cell level. However, nitrocefin was not retained within the periplasm and was, thus, unable to distinguish resistant from susceptible cells using microscopy. It has to be noted that also fluorogenic β-lactamase substrates like CCF2-AM are available, but these substrates are much more expensive and their application is mainly limited to eukaryotic reporter gene assays.
Comparison of culture-independent techniques applied for the screening of antibiotic resistance
Simultaneous species identification
Narrow, type-specific (e.g. TEM)
Narrow, type-specific (e.g. TEM)
Transcription is no correlate for efficient expression
Specific antibodies required
Not suitable on the single cell level
Fluorescence in situ hybridization and immunofluorescence tests represent promising and affordable tools for susceptibility testing on the single cell level. They combine the speed of other rapid and culture-independent methods with the ability of the cultural methods to obtain functional information and, furthermore, have the potential for simultaneous species identification.
AR, JAH and SAD designed the study. AR carried out the experiments and analyzed the data. AR, JAH and SAD drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data supporting the conclusions of this article are incorporated in this manuscript and in its supplementary information files.
This work was supported by a grant of the German Federal Ministry of Education and Research and was executed within the framework of the project ZooGloW (FKZ 13N12697). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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