Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification

Introduction

Enterococci are found in the gastrointestinal tract of animals and humans as well as in soil, water and food contaminated with feces. They are used as a bacterial indicator of fecal contamination in water and food. The concentration of enterococci in human stool ranged from 10⁵–10⁸ colony forming unit (CFU)/gram (Gelsomino et al., 2003; Kleessen, Bezirtzoglou & Mättö, 2000). In addition, they are also one of the most prevalent nosocomial pathogens, causing various infections such as urinary tract infections, endocarditis and bacteremia. In the treatment of enterococcal infection, ampicillin remains the antibiotic of choice, while vancomycin is utilized in cases of ampicillin resistance (Kristich, Rice & Arias, 2014). The increased usage of vancomycin has led to a rise in the number of vancomycin-resistant enterococci (VRE) infections. VRE was found to be 8.10% prevalent throughout Asia. The Enterococcus faecium showed higher resistance to vancomycin than E. faecalis (22.4% vs. 3.7%) (Shrestha et al., 2021). As shown in a survey from China, Enterococcus spp. was the most common pathogen in nosocomial bloodstream infections, of which 74% were E. faecium and 20% were E. faecalis. Furthermore, it was associated with high mortality rate of up to 24% (Zhang et al., 2017). The National Antimicrobial Resistance Surveillance Centre of Thailand revealed that VRE infection rate of E. faecium has climbed from 0.7% in 2012 to 6.9% in 2020, whereas VRE in E. faecalis has remained at 0.3% for the past ten years (National Antimicrobial Resistance Surveillance Center Thailand, 2020). The vanA and vanB types of VRE are the most common, especially in E. faecium isolates (Hollenbeck & Rice, 2012). The VRE is associated with high-mortality enterococcal bloodstream infections (Chiang et al., 2017). Most hospitals screen for VRE carriers from rectal swab samples, in order to avoid enterococcal bloodstream infections, prevent the spread of nosocomial infection and establish a successful treatment strategies.

The conventional identification of VRE is generally based on culture, biochemical tests, disk diffusion and determination of minimal inhibitory concentration, as well as genotypic methods (Jenkins & Schuetz, 2012). However, all these methods are time-consuming. Molecular techniques were developed to identify the species of Enterococcus several decades ago (Dutka-Malen, Evers & Courvalin, 1995). Although the polymerase chain reaction (PCR) method is highly sensitive and specific, it has been limited to well-equipped facilities due to the thermocycling requirements. The isothermal amplification techniques have been developed to overcome this constraint, such as recombinase polymerase amplification (RPA), which was initially reported in 2006 (Piepenburg et al., 2006). RPA technique is based on the functions of three proteins comprising recombinase, recombinase loading factor and single-stranded binding (SSB) protein. The nucleoprotein complex is formed by the interaction of an oligonucleotide primer and a recombinase. The recombinase loading factor assists in the combination. The complex searches for the homologous sequences on DNA template and replaces them in the duplex DNA. The formation of primer-DNA complex is stabilized by SSB protein (Yonesaki & Minagawa, 1985). DNA polymerase initiates the amplification at the end of the primer and finally results in the amount of amplified DNA (Piepenburg et al., 2006). The RPA reaction is enzymatically processed and does not require thermal cycling. The RPA technique can detect very small amount of target DNA molecules. It only requires a low constant temperature (25–42 °C) and a 20 min turnaround time. Moreover, the RPA product can be detected by using a variety of methods, such as agarose gel electrophoresis (AGE), real-time quantitative fluorescence or electrochemical detection, etc. Alternatively, the lateral-flow strips (LF) can be used to visualize the amplicons in 5 min, which is highly practical. The LF strip is a simple tool for qualitative testing, based on sandwiches assay by adding a specific probe into RPA reaction solution. The RPA products would be detected by using specific antibody to the pathogen, tagged with gold nanoparticles which is applied on a paper strip. The results of LF are visible and can be read in 5–10 min (Jauset-Rubio et al., 2016).

Therefore, we developed a rapid and sensitive RPA-LF method for detecting E. faecium and its important vancomycin-resistance genes (vanA and vanB) in positive blood-culture, stool and rectal-swab samples.

Materials & Methods

Results

Determination of the detection limit of PCR-AGE, RPA-AGE and RPA-LF method

The lower limit of the PCR-AGE method for detection of E. faecium, and of vanA- and vanB-carrying VRE were 10⁸, 10⁶ and 10⁷ CFU/mL, respectively, while the detection limit of the RPA-LF and RPA-AGE methods were 10⁷, 10⁵ and 10⁶ CFU/mL, respectively (Fig. 1). The detection limit of RPA method was 10 times lower than that of the PCR-AGE method.

Discussion

E. faecium is microorganism present in the intestines of humans and animals that can cause various infections including bacteremia, which can lead to considerable morbidity and mortality. In the United States, around 83 percent of E. faecium were vancomycin resistant in bloodstream infections patients (Weiner et al., 2016). VRE infections in the bloodstream had a greater mortality rate than those of vancomycin-susceptible Enterococcus (DiazGranados et al., 2005). VRE can survive in various adverse environments such as a wide range of temperature (10–45 °C) and pH (pH 4.6–9.9), 40% bile salts and 6.5% NaCl (Vanden Berghe, De Winter & De Vuyst, 2006; Foulquie Moreno et al., 2006), particularly in healthcare settings. VRE-carrier screening using stool or rectal swab sample is recommended by Hospital Infection Society and Infection Control Nurses Association to reduce the spread of VRE and related infections in hospitals (Cookson et al., 2006). Development of reliable and sensitive methods for VRE detection is therefore of paramount importance. The RPA-LF method has been adapted to detect many pathogens which requires minimal equipment and a quick turnaround time of 30 min. It has been applied for detection of pathogens in various samples, such as blood (Srisrattakarn et al., 2020), plasma (Qi et al., 2018), foodstuffs (Du et al., 2018a; Du et al., 2018b), stool (Crannell et al., 2014a) and environmental samples such as plant (Londono, Harmon & Polston, 2016), soil and water (Saxena et al., 2019). To the best of our knowledge, this is the first report of the application of the RPA-LF method for direct detection of E . faecium and the vanA and vanB genes in positive blood-culture bottles and stool/rectal-swab samples, facilitating immediate medical intervention.

The optimal conditions of the RPA reaction for detection of the three targets (E . faecium and vanA and vanB genes) were 37 °C for 20 min. Several investigations have shown that the RPA-LF can function well at temperatures ranging from 25–45 °C (Lu et al., 2021; Peng et al., 2019) and the faint band was visible in 5 min (Du et al., 2018a; Peng et al., 2019; Srisrattakarn et al., 2020; Wu et al., 2020). Similarly, our study found that the RPA worked effectively at a wide temperature range (25–42 °C) and incubation times of 5-30 min could generate a visual signal on the test lines. We chose 37 °C because all reactions yielded strong bands at the test lines and because low-resource settings usually have equipment allowing incubation at this temperature. The RPA reaction was incubated for 20 min to prevent false positive results due to unexpected amplification in long time of reaction (Piepenburg et al., 2006). Note that the RPA product was diluted 1:100 with buffer to reduce nonspecific binding of crowding agent and proteins in RPA reactions with the antibodies on a lateral flow strip, and the LF test must be read within 5 min to prevent a false positive result (Rosser et al., 2015; Saldarriaga et al., 2016; Wu et al., 2016).

The RPA-LF method for detection of the three genes was 10 times more sensitive (lower detection limit) than the conventional PCR method, whereas the detection limits of the RPA-LF and RPA-AGE methods were equal. The RPA reaction has higher sensitivity than the PCR method because the RPA primer is designed according to the TwistAmp^® kit formulations which indicated the support to the RPA reaction. The RPA reactions contain a high level of ATP-burning recombinase that the fuel is consumed typically within around 25-30 min. RPA primers (30–35 bp) are longer than the typical conventional PCR primers for the amplification to give a DNA product of 100–500 bp (TwistDx Limited, 2018). The primers length of greater than 28 bp bind strongly to the recombinase protein. These complex searches for the homologous sequences in the double-stranded DNA and invading the DNA template. The recombinase-primer complex stimulates the ATP hydrolysis in RPA reaction. The complex will split when the ATP is hydrolyzed, and the recombinase will bind to the primer to begin the next reaction immediately (Piepenburg et al., 2006). Therefore, the RPA has higher sensitivity and gives a faster result than the conventional PCR reaction. The detection limit of RPA in our study was lower than the conventional PCR, which was consistent with the study of Wang et al. (2016).

The performance of the RPA-LF for detection of pathogens in both blood culture and rectal swab/stool samples showed 100% sensitivity and 100% specificity with no cross reactions. The method has been successful for direct detection of E . faecium and vanA or vanB genes in positive blood-culture samples. Several studies showed that some components of blood, mainly heme (Akane et al., 1994), leukocyte DNA (Morata, Queipo-Ortuno & Colmenero, 1998), immunoglobulin G in plasma (Al-Soud, Jonsson & Radstrom, 2000), and various inhibitors in the environment (Jiang et al., 2005) are major inhibitors of the PCR reaction. The RPA-LF technique was shown to be successful even in serum, heparin and hemoglobin (Kersting et al., 2014). However, some technical problems of the RPA-LF reaction were encountered during the study, when tested with fecal samples directly. It is known that fecal samples contain various substances that influence the reactions (Schrader et al., 2012). Additionally, the culture medium for bacterial enrichment or the solution for sample extraction may contain inhibitors. In this study, the bile esculin inhibited the RPA-LF reaction resulting in negative results (Fig. S3). The cetyltrimethyl ammonium bromide in DNA extraction buffer was reported to have a strong inhibitory effect on RPA reactions (Valasevich & Schneider, 2017). Therefore, sample preparation steps are crucial to reduce those substances. In our study, the faecal debris and medium broth were eliminated as feasible after the incubation period. Then the suspected microbial suspension was used as sample for the RPA-LF reaction, which yielded satisfactory results despite the lack of DNA extraction. The RPA-LF reaction can tolerate crude samples with minimal sample preparation steps (Silva et al., 2018) and in the presence of many inhibitors (Kersting et al., 2014). In comparison to the results obtained from conventional PCR method and the developed RPA method of Yin et al. (2017), the RPA method has a high sensitivity and specificity. However, development of RPA for detection of Burkholderia pseudomallei in blood samples (Peng et al., 2019) and detection of group B streptococci in vaginal/anal samples (Daher et al., 2014) were compared to real-time PCR method, the latter report showed that RPA assay had 96 and 100% of sensitivity and specificity, respectively.

In our detection limit study, the result of conventional PCR reaction was negative, while that of the RPA reaction was positive. This occurred when there was low number of bacteria in the sample which affected the capability of the detection method. The number of bacteria in each sample in this study was at least 10⁸ CFU/ mL, which could be detected from both RPA and conventional PCR. As a result, the calculated values of sensitivity and specificity were 100%. However, the real-time PCR method should be used as a reference method to support in the further research, particularly for the detection of samples containing a low number of bacteria.

The RPA method uses simple incubation conditions and requires common laboratory equipment such as a heat block and water bath. Moreover, the RPA processes for DNA amplification can be incubated at the body temperature (Crannell, Rohrman & Richards-Kortum, 2014b). While the conventional or real-time PCR methods and GeneXpert need special equipment such as thermocycle machine or GeneXpert instrument (Cepheid, Sunnyvale, CA, USA). RPA method displays a faster time-to-result (<20 min) than the PCR method (>1.5 hr) and the GeneXpert (∼45 min) (Moore & Jaykus, 2017; Cepheid, Sunnyvale, CA, USA). These are the main benefits of the RPA for VRE detection at the point of care setting. Furthermore, one of the most important factors to consider is the cost. The RPA platform is more cost-effective (∼$3.70) compared with the costs of conventional PCR (∼$2.60) and real-time PCR (∼$25) (Al-Siyabi et al., 2013; Richards et al., 2019; Zhang et al., 2014). However, the volume of RPA reaction can be reduced to 12.5 µL leading to decrease in the cost of RPA-LF method to ∼$2.28 per test (∼$0.83 for RPA reaction and ∼$1.45 for the haft of commercial LF strip), which was adopted in this study.

One limitation of this study is the small number of clinical samples that we used. VRE bloodstream infections are rare in the hospital, limiting the availability of such samples. Therefore, additional tests were carried out using spiked samples. However, additional clinical samples for testing are still needed, especially those of the vanB-type VRE, which are particularly uncommon in our area. In addition, E. faecalis can cause a variety of nosocomial infections such as urinary tract infections but the prevalence of vancomycin-resistant E. faecalis (0.3%) was lower than E. faecium (6.9%) in our area (National Antimicrobial Resistance Surveillance Center Thailand, 2020). Therefore, the method for detection of E. faecalis should be further studied. Another limitation in this study was a shortage supply of TwistAmp nfo kit from the manufacturer, forcing us to test a limited number of samples, especially for the vanB gene and lacking of the RPA-LF test for E. faecalis.

Conclusions

The RPA-LF method is rapid (within 30 min), user-friendly test for detecting E. faecium and vanA or vanB genes. It exhibits high sensitivity and specificity. This is a suitable candidate for use in low-resource laboratories and would also be useful for infection-control purposes to prevent the spread of VRE in hospitals.

Supplemental Information