A TaqMan-based multiplex real-time PCR assay for the rapid detection of tigecycline resistance genes from bacteria, faeces and environmental samples

Tigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant bacteria. Recently, novel tigecycline resistance genes tet(X3) and tet(X4) have been reported, which pose a great challenge to human health and food security. The current study aimed to establish a TaqMan-based real-time PCR assay for the rapid detection of the tigecycline-resistant genes tet(X3) and tet(X4). No false-positive result was found, and the results of the TaqMan-based real-time PCR assay showed 100% concordance with the results of the sequencing analyses. This proposed method can detect the two genes at the level of 1 × 102 copies/μL, and the whole process is completed within an hour, allowing rapid screening of tet(X3) and tet(X4) genes in cultured bacteria, faeces, and soil samples. Taken together, the TaqMan-based real-time PCR method established in this study is rapid, sensitive, specific, and is capable of detecting the two genes not only in bacteria, but also in environmental samples.


Background
With the prevalence of antimicrobial resistance (AMR), only a few antibiotics are available to treat severe infections caused by extensively drug-resistant (XDR) bacteria, which poses a great challenge to human health and food security. Tigecycline and colistin are last-resort drugs to treat infections caused by carbapenem-resistance Enterobacteriaceae [1]. The World Health Organization (WHO) classified the two antibiotics as critically important antimicrobials, and their usage should be severely restricted (http://www.who. int/foodsafety/publications/antimicrobials-fifth/en/) [2]. Since the recent reports of colistin resistance genes (mcr), the clinical application of colistin has become more limited [2,3], turning tigecycline into the ultimate treatment option.
To date, both genes have been identified in humans, animals, meat, and environmental samples [4,[6][7][8]. In three representative provinces of China, tet(X3) and tet(X4) genes have been identified in animals and meat for consumption at a high proportion of 43.3% [4], indicating the wide transmission of tigecycline resistance. Recent studies showed that the presence of tet(X3) and tet(X4) genes can significantly increase the resistance to tigecycline, and the construction of a tet(X4)-containing bacterial strain, namely, Escherichia coli JM109 + pBAD24-tet(X4), increases the MIC value of tigecycline by 64-fold compared with the original host strain [8].
Therefore, it is necessary to establish an efficient method for simultaneous screening the tigecycline-resistant genes tet(X3) and tet(X4) in different samples. Nowadays, realtime PCR assays are widely used in laboratories domestically and overseas. Compared to conventional PCR, real-time PCR has superior sensitivity, reproducibility, precision, and high throughput capability [9]. In this study, we designed a rapid, sensitive TaqMan-based multiplex real-time PCR assay for the specific detection of the tigecycline resistance genes tet(X3) and tet(X4), and further evaluated using cultured bacteria, faeces and soil samples.

Primers and probes
The results of the NCBI Primer BLAST module indicated that no genes other than tet(X3) and tet(X4) genes matched the primer sequences designed in this study. Similarly, the results of conventional and real-time PCR also indicated the high specificity of primers and probes (Fig. 1).
Linear standard curves for real-time PCR are shown in Fig. 2. The amplification efficiencies were calculated using the formula E = 10 (− 1/slope) − 1 [10]. R 2 values were 0.995 and 0.999, respectively, and efficiency was 90.58 and 97.12% for the tet(X3) and tet(X4) genes, respectively. The sensitivity of analysis was linear within the dynamic range of 9 dilutions. These results reveal that these two real-time PCR tests are accurate for quantitative detection of tet(X3) and tet(X4) genes.

Specificity and sensitivity evaluation
To confirm the specificity of the assay, two E. coli DH5α strains carrying tet(X3) or tet(X4) were used as the positive control, while E. coli ATCC25922 and three E. coli DH5α strains containing tet(X), tet(X1) or tet(X2), respectively, were used as the negative control. Each sample was tested three times independently (n = 3). The results of TaqMan-based real-time PCR were 100% concordance with the results of conventional PCR (Table 1 & Fig. 1), which proved that the two primer sets and probes were highly specific for their target gene.
To further validate the method, genetic DNA extracted from bacteria, faeces and soil samples (three independent technical replicates) was selected to conduct the real-time PCR assay for screening. In this study, some E. coli and A. baumannii isolates of animal origin were selected for verification, including 4 tet(X3) positive strains and 8 tet(X4) positive strains ( Table 1). The CT ranges were 19.05-21.06, 13.42-21.05 for tet(X3), tet(X4) genes (Table 1). The results of real-time PCR and previous sequencing analyses were completely consistent, showing high specificity of the method. Moreover, a total of 24 faeces and soil samples from chickens, pigs, and cattle were collected for further evaluation. We were able to detect the two genes in metagenomes extracted from 19 faeces and soil samples, and the relative abundance was normalized using 16S rRNA (gene copies/10 6 copies of the 16S rRNA gene) [11,12]. The real-time PCR assay showed that the normalized copies range from 10 1 to 10 5 for genes tet(X3) and tet(X4) (Fig. 3).

Discussion
Since the first discovery of the tet(X3) and tet(X4) genes, these two plasmid-mediated tigecycline resistance genes have been widely reported, suggesting that they are spreading at an alarming rate. It is noteworthy that the tet(X3) and tet(X4) genes have been identified not only in humans and animals, but also in the environment [4,13,14]. There    . 3 Detection of tet(X3) and tet(X4) genes in faeces and soil samples. The relative abundance of tet(X3) and tet(X4) genes (copies /per 1,000,000 copies of 16S rRNA) in the soil and faeces samples is a pressing need to establish a fast screening assay for tigecycline resistance genes. So far, there are three reports of fast screening of tet(X3) and tet(X4), multiplex PCR methods, tetracycline inactivation method, and SYBR Green based real-time PCR, all three methods have approved the effectiveness of methods [15][16][17]. Compared to traditional detection methods, like conventional PCR and phenotypic method, real-time PCR assays are more sensitive, specific, time-efficient, and labour-saving [18]. Besides, real-time PCR methods can detect genes in different type of samples other than bacteria. Lately, Fu et al. reported a SYBR Green-based real-time PCR assay for rapid detection of tet(X) variants, with a detection limit range from 10 2 to 10 5 , 1-10 3 per 10 6 copies of 16S rRNA for tet(X3) and tet(X4), respectively. To date, the method based on realtime PCR using TaqMan probe has not been previously proposed.
In this study, we developed a TaqMan-based multiplex real-time PCR assay for the detection of the tet(X3) and tet(X4) genes. Both tet(X3) and tet(X4) genes have been successfully identified not only in bacteria isolates, but also directly from faeces and soil samples, with a minimum of 1 copy per 10 5 copies of 16S rRNA and a maximum of 10 4 copies per 10 5 copies of 16S rRNA. Besides, we used constructed E. coli DH5α-tet(X) strains as positive control and bacteria isolates of animal origin to evaluate the specificity of the method. The E. coli ATCC25922 was used as negative control. In our method, only specific amplicons can be bonded by TaqMan probes, which is different from the SYBR-Green dye. The SYBR-Green can bind any double strand DNA fragments in the PCR reaction without any specificity, so melting curve analysis will be necessary to identify the single peak for the PCR reaction. According to the Fig. 1, our method has high specificity, whereas other tet(X) genes couldn't be amplified. A limitation of the proposed method is that these genes cannot be detected simultaneously in a single reaction. However, there are no reports of the co-existence of these two genes. Because tet(X3) and tet(X4) genes are usually accompanied with MDR [9], different combinations of such detection methods are flexible and convenient.
Although tigecycline usage has never been approved in animal husbandry, tetracyclines have been widely used in China and many other countries. The total consumption of tetracyclines reached 13,666 tons in 2018 in China, which may provide ongoing selective pressure for the production of tigecycline resistance genes. Many studies have shown that tet(X4) can be captured by a range of mobile elements circulating among bacterial strains [4,[6][7][8], importantly, with the international trade of food-producing animals and their derivatives, a novel antibacterial mechanism may appear. It is of great importance to monitor and eradicate these genes, especially in countries with high tetracycline consumption.

Conclusion
Overall, we developed a TaqMan-based multiplex realtime PCR method in this study for the rapid detection of tigecycline resistance genes, tet(X3) and tet(X4). This assay can be widely applied to all laboratories equipped with a qPCR machine, and the whole process could be completed within an hour. It is highly sensitive and specific, and can detect and quantify tet(X3) and tet(X4) genes accurately in cultured bacteria isolates, faeces and environmental samples.

Bacteria strains and environmental samples
All the E. coli and A. baumannii strains from animal origin used in this study (Table 1) were collected from three poultry farm in Shandong Province, and were identified by conventional PCR and MALDI-TOF before. Five tet(X) variant genes were cloned into the PMD-19 T vector (Takara Bio, Kusatsu, Japan), and then transferred into the DH5α cell, including DH5α-tet(X), DH5αtet(X1), DH5α-tet(X2), DH5α-tet(X3) and DH5α-tet(X4) ( Table 1). A total of 24 faeces and soil samples collected from chickens, pigs, and cattle farms from Sichuan Province were then used for further evaluation (Fig. 1).

DNA extraction
Bacteria were incubated at 37°C in Brain Heart Infusion broth with agitation at 200 rpm to achieve enough colonies for DNA extraction. Hipure Bacterial DNA Kit (Magen, Guangdong, China) were used to extract bacterial genome according to the manufacturer's instruction. The DNeasy PowerSoil (Qiagen, Hilden, Germany) was used to extract metagenomic DNA from faeces and soil.

Primer and probe design
The nucleotide sequences of tet(X3) and tet(X4) were obtained from GenBank. The specific real-time PCR primers

Specificity and sensitivity tests
To evaluate the specificity of the proposed method, DH5α strains containing tet(X), tet(X1), tet(X2), tet(X3) and tet(X4), respectively, were used to conduct the realtime PCR assay (Table 1). Genomic DNA extracted from bacteria, faeces and soil samples from different origin was then used to further validate the specificity and sensitivity of the method.