- Methodology article
- Open Access
Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzyme
© Franz et al. 2016
Received: 22 September 2015
Accepted: 9 March 2016
Published: 18 March 2016
The Erratum to this article has been published in BMC Microbiology 2016 16:219
To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology.
Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced.
These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.
Bacterial and fungal infections are one of the major threats to human health. Among others, increasing resistances of pathogenic microorganisms against frequently used antibiotics are raising the number of dangerous infections every year . Hence, a fast and reliable identification system to achieve an effective and specific medical treatment is essential [2, 3]. In the last decades, procedures based on morphological, physiological and biochemical analyses were progressively replaced by more sensitive PCR amplifications of pathogen-specific gene sequences. These target sequences exhibit patterns of characteristic point mutations, allowing a robust species-specific verification of an organism [4, 5].
In this study, we present a new and highly specific target sequence for genotyping bacterial and eukaryotic pathogens. The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) represents an essential and ubiquitous tRNA maturation activity found in all kingdoms of life. The enzyme catalyzes the posttranscriptional addition of the CCA triplet to the 3′-end of tRNAs, generating the site of aminoacylation [6–8]. Even in organisms where the CCA triplet is encoded in the tRNA genes, like E. coli or certain Bacillus species, a CCA-adding enzyme is encoded in the genome, as it is also required for the maintenance and repair of the CCA-ends in the tRNA pool [9–11].
Besides V. cholerae, other Vibrio species represent highly pathogenic germs as well. V. alginolyticus, V. parahaemolyticus and V. vulnificus are gram-negative, halophilic bacteria that are frequently found in estuarine, coastal and other nutrient-rich waters around the world [16–22]. They are frequently detected in seafood like oysters, shrimps or soft-shell clams. Consequently, they are associated with gastroenteritis and septicemia as well as wound infections due to direct contact with contaminated water [23–31]. Studies in the United States show a correlation between V. parahaemolyticus counts and the number of zooplankton populations, which are increasing during the summer months, when the incidence for Vibrio infections is high [32–34]. In cooler seasons, Vibrio species only occur in sediments or shellfish before they start to proliferate again at warmer temperatures with one of the fastest bacterial growth rates [32, 35–38]. Accordingly, increased surface temperatures of lakes and seas correlate with an elevated infection incidence [39–43].
In addition to these examples of closely related prokaryotic pathogens, we have included several species of Aspergillus, representing a eukaryotic disease-causing fungus. The most prominent pathogenic species of this mold genus are A. fumigatus, A. niger and A. terreus, triggering life-threatening invasive infections which are a leading cause of mortality in immunocompromised patients [44–49]. A. fumigatus is the main causative agent of invasive aspergillosis that affects skin, ears, paranasal sinuses or the lung, resulting in a mortality rate of nearly 100 % in untreated patients . Infections with A. niger and A. terreus also frequently cause serious aspergillosis with comparable mortality rates . These infections represent a worldwide health threat, as several studies show a ubiquitous presence of Aspergillus species. In addition to soil samples and internal building walls, they can also be isolated from clothes, shower heads, dusty air conditioners and hospital plants, resulting in a constantly high infection incidence in immunocompromised patients [48, 50].
Thus, a fast and reliable identification of these pathogens is very important for an immediate and successful medical treatment of such infections. Using the highly variable gene sequence encoding the flexible loop element of CCA-adding enzymes, we show that very closely related Vibrio and Aspergillus species can be discriminated at a much higher fidelity compared to approaches focusing on standard gene sequences in diagnostics.
One of the essential elements in the catalytic core of CCA-adding enzymes consists of a flexible loop region located between the conserved sequence motifs A and B (Fig. 1a). In contrast to other elements of the catalytic core, the amino acid composition of the loop reveals a remarkable sequence diversity. Hoffmeier et al. could show that this variety in the loop sequence correlates with the evolutionary distance between the corresponding organisms . According to detailed sequence alignments, distant organisms exhibit great differences in their loop sequence, whereas the sequences of closely related organisms are more similar and can be summarized into loop families. Yet, the loop-encoding DNA sequences can be used for a reliable genotyping and are better suited for a robust species-specific identification than 16S rRNA analysis, a standard tool to identify and distinguish certain pathogenic bacteria .
Discriminatory potential of the loop-encoding DNA sequence compared to 16S (Vibrio) or 18S rRNA gene (Aspergillus)
16S rRNA gene
CCA-adding enzyme loop
Number of specific mutations
Number of specific mutations
18S rRNA gene
In addition to these bacterial species, representatives of pathogenic fungi were investigated as well. The corresponding gene sequences (18S rRNA gene, loop region of the CCA-adding enzyme gene) were analyzed in three closely related Aspergillus species, using genomic DNA of A. fumigatus, A. niger (ATCC 6275) and A. terreus (ATCC 10690). In all investigated 16S or 18S rRNA gene sequences, a discriminatory potential represented by percentage sequence differences from 0.1 to 1.1 % (V. parahaemolyticus, V. alginolyticus) and 0.1 to 0.5 (all tested Aspergillus species) was observed. Only the 16S rRNA gene of V. vulnificus exhibits a higher sequence deviation with 42 point mutations and deletions, leading to a ratio of 4.9 %. In all cases, a much higher sequence difference was observed for the gene region encoding the flexible loop of the corresponding CCA-adding enzymes. Here, mutation ratios between 10.7 and 13.3 % (V. alginolyticus, V. parahaemolyticus) and 11.1 and 15.9 % (Aspergillus species) were identified, whereas the discriminatory potential of the loop sequence from V. vulnificus reaches 28.0 % (Table 1). In this case, the comparative potential (represented by the ratio between specific mutations (in %) in the loop and the 16S rRNA sequences) is 5.7 times higher than that of the 16S rRNA analysis. For the other investigated organisms, the discriminatory potential is also higher for the loop sequence, ranging from a factor of 12.1 to 35.8. In the case of V. parahaemolyticus and A. niger, the first 850 nucleotides of the corresponding rRNA genes revealed only one characteristic point mutation, whereas the loop sequence presents 8 and 10 point mutations in a sequence of 75 and 63 nucleotides, respectively. Thus, the distinction potential is increased by factors 107.0 and 159.0 in these species.
Intraspecific sequence similarities of selected Vibrio and Aspergillus species
Number of strains
min. point mutations
max. point mutations
Usually, patient samples do not only contain DNA from the pathogen but carry an excess amount of endogenous human genomic DNA. As the human genome also encodes for a related CCA-adding enzyme, it is important that the genotyping procedure strongly discriminates against such endogenous contaminations. Hence, 0.1 ng of Vibrio DNA was mixed with an increasing amount of human genomic DNA, leading to a 1,000-fold excess of human gene sequences. With such contaminated material, a multiplex PCR containing the above mentioned primer combinations was performed and the reaction products were separated on an agarose gel (Fig. 3b). The resulting fluorescent signals indicate that the excess of human DNA has only a minor effect on the amplification efficiency of the Vibrio sequences, while no unspecific reaction products resulting from hybridization to the human DNA were observed. Nevertheless, we have designed a human-specific blocking oligonucleotide carrying a C3 spacer at the 3′ position that efficiently inhibits the amplification of the human loop-encoding sequence, while it does not interfere with PCR-based detection of bacterial sequences (Additional file 1: Figure S1) . Hence, if needed, this blocking oligonucleotide can be added to the PCR reaction in order to enhance the detection specificity.
Taken together, these results confirm that the DNA sequence encoding the flexible loop from CCA-adding enzymes represents a unique and highly specific amplification target that allows a fast and reliable genotyping of different Vibrio or Aspergillus infections.
Infections caused by pathogenic microorganisms represent a widespread threat of human health. Especially the incidences of foodborne diseases and invasive fungal infections caused by Vibrio and Aspergillus species are increasing every year. Representatives of these two genera are able to thrive on a broad spectrum of substrates and environmental conditions, leading to their world-wide distribution that is even enhanced by climate changes. Especially the three non-cholera Vibrio species V. alginolyticus, V. parahaemolyticus and V. vulnificus, all thriving in coastal and estuarine waters, are benefiting from global warming and increasing surface temperatures of lakes and seas all over the world [39, 42, 43, 54, 55]. Hence, the development of fast and reliable identification methods for such human pathogens is urgently needed. Since many microbiological analyses were replaced by faster and more sensitive PCR methods, many specific gene sequences were identified that allow for a distinction between closely related species of the same genus. For such species-specific identification, the analysis of 16S and 18S rRNA gene sequences represents a widely used standard approach [2, 51, 56–58]. However, in the case of V. alginolyticus, V. parahaemolyticus and V. vulnificus as well as A. fumigatus, A. niger and A. terreus, the distinctive potential of the corresponding ribosomal DNA sequences is rather low, ranging from 0.1 to 4.9 % (Table 1). The high similarities in the 16S rRNA gene sequence were already described by Kwok et al., who observed a sequence identity of 99 % between V. alginolyticus and V. parahaemolyticus and a 95 % between V. alginolyticus and V. vulnificus, as well as between V. parahaemolyticus and V. vulnificus . Likewise, other studies revealed that rRNA sequence homologies between V. parahaemolyticus and related species are so high that this gene is not suited for identification approaches [60, 61].
To overcome these limitations, more specific template sequences for reliable genotyping were established in the last years. Especially for the discrimination of Vibrio species, pathogenicity markers like thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) are analyzed [62–65]. A very useful target sequence to identify different Vibrio species is the toxR gene, a toxicity-related transcriptional regulator originally found in V. cholerae [5, 66, 67]. Combined in multiplex PCR or multi locus sequence typing approaches, these Vibrio-specific target genes are powerful tools for a solid identification procedure. However, they are restricted to a small pool of organisms within a genus, showing no distribution in other human-relevant pathogens. Studies on more ubiquitous gene sequences, like the B subunit of the bacterial DNA gyrase (gyrB), the hsp60 or the rpoB gene, revealed differences ranging from 3.2 to 20.0 % between V. alginolyticus, V. parahaemolyticus and V. vulnificus [59, 68, 69]. In 2010, Pascual et al. published a multi locus sequence analysis of several closely related Vibrio species, calculating the intra- and interspecific sequence similarities of the most promising genotyping sequences. Intraspecific sequence similarities were ranging from 98.8–100 % (16S rRNA gene), 92.7–100 % (recA), 93.7–100 % (pyrH), 95.6–100 % (rpoD), 86.8–100 % (gyrB), 85.6–100 % (rctB) and 77.2–100 % (toxR), with interspecific similarities of 97.6–99.9 % (16S rRNA gene), 87.9–99.9 % (recA), 86.4–97.8 % (pyrH), 79.1–96.0 % (rpoD), 83.1–99.5 % (gyrB), 74.3–92.7 % (rctB) and 33.8–72.5 % (toxR) . According to these values, the best taxonomic resolution is given by the sequences of rctB, rpoD and toxR.
Taken together, these classification parameters indicate that the flexible loop region of CCA-adding enzymes has an identification potential similar to that of established genetic Vibrio markers, although its sequence with a length of 75 bp is much shorter than that of the main discriminatory regions of toxR (477 bp), rctB (591 bp) and rpoD (780) . Furthermore, while toxR and rctB markers can only be used for Vibrio genotyping, CCA-adding enzyme genes with varying flexible loop regions are found in all bacteria and eukaryotes, indicating that the loop sequence has the potential as a genotyping marker for a wide range of different pathogens .
This is further supported by our findings concerning the genotyping of individual Aspergillus species. Here, the discriminatory potential of the loop sequences of A. fumigatus, A. niger and A. terreus is even higher, with interspecific similarities between 74.6 and 79.4 % compared to 99.2–99.5 % in the 18S rRNA gene sequences (Table 1, Fig. 1b). In addition, no intraspecific point mutations were observed (Table 2), leading to an excellent taxonomic resolution. Yet, 18S rRNA sequence analysis is frequently used for Aspergillus genotyping [2, 70, 71]. Alternatively, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism PCR (RFLP-PCR) are used, where the internal transcribed spacer regions between 18S, 5.8S and 28S rRNA genes are investigated [50, 72–74]. Both methods are suitable for a reliable identification of Aspergillus species, but the correct interpretation of the resulting complex DNA fragment patterns is difficult . Furthermore, these methods cannot be used for analyzing several species within a single multiplex PCR, as it was done in this study. In contrast, the flexible loop-encoding region of the gene for CCA-adding enzyme is much better suited for identification of pathogenic microorganisms (in single or multiplex PCR), fulfilling the criteria for a successful application in genotyping . First, as the CCA-adding enzyme represents an essential protein, the corresponding gene is ubiquitously distributed in the kingdoms of Bacteria and Eukaryotes [75, 76]. Second, to allow a specific sequence analysis, the target gene must be unique within a genome, without closely related paralogs. Bacterial and eukaryotic CCA-adding enzymes are closely related to bacterial poly(A) polymerases, sharing the same conserved core motifs [77, 78]. However, the flexible loop element of the poly(A) polymerase has a very unique sequence composition, showing no similarity to the loop sequence of CCA-adding enzymes . Furthermore, in several bacterial species, the CCA-adding activity is shared between two closely related enzymes, where the first catalyzes the addition of two C residues (CC-adding enzyme), while the second one adds the terminal A (A-adding enzyme) [79–81]. Yet, such a gene constellation still allows for a specific species identification, as CC-adding enzymes have lost the loop region due to a deletion of the corresponding coding region . In contrast, A-adding enzymes carry a loop sequence. However, this sequence shows the same discriminatory potential as in CCA-adding enzymes, so that the loop region in A-adding enzymes can also be used for genotyping. Third, the gene sequence has to be long enough to contain sufficient phylogenetic information, and, on the other hand, short enough for fast sequence analysis using a minimal set of primers. With a length of 63–75 bp, as found in Vibrio and Aspergillus species as well as in other organisms, the loop-encoding region is indeed very short. Nevertheless, our data indicate that it contains a high number of unique and characteristic sequence differences for a species-specific amplification and identification.
Taken together, the DNA sequence encoding the flexible loop region of CCA-adding enzymes shows all features required for a highly discriminating gene marker in pathogenic microorganisms. This is supported by the presented results, where closely related pro- and eukaryotic pathogens could be easily discriminated, in contrast to most of the established diagnostic markers. Although in the case of Aspergillus, no loop sequence information of type strains is available, the data clearly show that this DNA marker is also very useful for eukaryotic pathogens. Combined with other sequences with high discriminatory potential, this new marker will contribute to a superb and highly reliable diagnostic procedure in infectology and systematic microbiology. Furthermore, the loop sequence can be a useful diagnostic tool in food hygiene analysis or in the detection of specific microbial species in so far unidentifiable metagenome DNA sequences.
Microbial strains and DNA extraction
Genomic DNA of V. alginolyticus (ATCC 17749), V. parahaemolyticus (ATCC 17802) and V. vulnificus (ATCC 27562) was obtained from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). A niger and A. terreus were obtained as freeze-dried cultures from the same institute. P. Zipfel (Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena, Germany) provided the genomic DNA of A. fumigatus. The freeze-dried cultures were handled as directed and dissolved in 40 % glycerol for storage at −20 °C. 50 ml Sabouraud dextrose broth were inoculated with 200 μl of the glycerol stock and incubated for 24 h at 37 °C and 200 rpm. After a centrifugation step, the pellet was resuspended in DNA extraction buffer containing 0.7 M NaCl, 0.1 M Tris–HCl (pH 7.5), 50 mM EDTA and 1 % SDS (w/v). Cells were lysed in a FastPrep®-24 system (MP Biomedicals, Heidelberg, Germany), using lysing matrix C at 6.0 m/s for 30 s. After a second centrifugation step, the supernatant was incubated for 5 min at room temperature and mixed with 5 ml 24:1 chloroform/isoamyl alcohol for 30 min on ice. The DNA in the supernatant was precipitated with 100 % ethanol. After centrifugation, the pellet was washed with 70 % ethanol, air dried, and dissolved in 50 μl double-distilled water. DNA concentrations were determined on a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Braunschweig, Germany).
Gene sequences of the individual CCA-adding enzymes were obtained from the National Center for Biotechnology Information (NCBI) database (Additional file 2: Table S1). The loop-encoding sequences were identified by aligning the corresponding DNA sequence between conserved core motifs A and B. 16S and 18S rRNA gene regions were sequenced on an ABI Prism 3700 automated sequencer (Amersham Biosciences). Accession numbers of further 16/18S rRNA genes used for interspecific alignments are summarized in Additional file 3: Table S2.
Sequences of primers used in this study
5′- GTAGGTGGCGCAGTTCGAG -3′
5′- GTGGGTGGAGCGGTAAGAG -3′
Degenerated forward primer for motif A region of V. alginolyticus/V. parahaemolyticus (sequence 1) and V. vulnificus (sequence 2)
5′- ACCAGTGTAACCAGAACCTGAC -3′
Reverse primer for loop sequence of V. alginolyticus
5′- TCCTCTTCTAGCGTTACGCTTG -3′
Reverse primer for loop sequence of V. parahaemolyticus
5′- CACATCTGGGGAGAAAAAGCAC -3′
Reverse primer for loop sequence of V. vulnificus
5′- GGGTGAGGGACAAGCTG -3′
5′- GGGTGCGGGACAAGCTG -3′
Degenerated forward primer for motif A region of A. fumigatus/A. niger (sequence 1) and A. terreus (sequence 2)
5′- CATCTTCTTCGGCGGTG -3′
Reverse primer for loop sequence of A. fumigatus
5′- CGTCTTCCTGGGCTGTTC -3′
Reverse primer for loop sequence of A. niger
5′- GGATTCCGACTATCATCCGTATAG -3′
Reverse primer for loop sequence of A. terreus
Standard and multiplex PCR amplifications
PCR was carried out in a volume of 20 μl of 20 mM Tris/HCl (pH 8.4), 50 mM KCl, 2 mM Mg Cl2, 150 μM dNTPs, 0.3 μM forward and reverse primers, 1 % DMSO, 1 U Taq DNA polymerase (NEB) and 0.1 to 3.0 ng of genomic DNA. An initial denaturation for 3 min at 96 °C was followed by 40 cycles of denaturation (96 °C, 60–90 s), primer annealing (53/59 °C, 60 s) and elongation (68 °C, 30/60 s). A final elongation was performed at 68 °C for 5 min. To improve the hybridization specificity in some of the reactions, a touch-down PCR was performed with temperature gradient for primer annealing from 60 °C to 53 °C within the first 10 cycles.
Detection of PCR products and imaging
PCR products were separated on an agarose gel and visualized by ethidium bromide staining. Fluorescent PCR products were detected by scanning the agarose gel in a Typhoon 9410 Variable Mode Imager using suitable laser-filter combinations. The absorption and emission maxima of the fluorescent labels are presented in Table 3. Resulting images were saved as tagged image files (tif).
We thank Peter Zipfel, Leibniz Institute for Natural Product Research and Infection Biology, Jena, for scientific discussions and for the generous gift of genomic DNA from A. fumigatus. Furthermore, we thank Frank Jühling, Leipzig University, for great help in bioinformatic analyses and discussions. This work was supported by the Else-Kröner-Fresenius-Stiftung (Az. 2011_A110).
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