Prevention of horizontal transfer of laboratory plasmids to environmental bacteria: comparison of the effectiveness of a few disinfection approaches to degrade DNA.

doi:10.21203/rs.3.rs-2609208/v1

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Prevention of horizontal transfer of laboratory plasmids to environmental bacteria: comparison of the effectiveness of a few disinfection approaches to degrade DNA.

https://doi.org/10.21203/rs.3.rs-2609208/v1

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published 13 Jul, 2023

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The routine work of any molecular biology laboratory includes the daily use of microorganisms, including strains of E. coli, transformed with a variety of plasmids expressing at least one antibiotic resistance gene (ARG).

To verify the effectiveness of disinfection methods on laboratory liquid waste, bacteria isolated from laboratory and research institute drains were identified by 16S ribosomal RNA sequencing and tested for the presence of an origin of replication and several ARGs frequently found in laboratory plasmids. Surprisingly, the origin of replication of Enterobacteriaceae plasmids was detected in strains of non-Enterobacteriaceae bacteria suggesting that interspecific transfer of laboratory plasmids had occurred.

Using quantitative Polymerase Chain Reaction, we determined the Decimal reduction value (D-value, expressed as concentration of disinfectant or length of physical treatment) of several decontamination methods for their DNA degradation effect on cultures of E. coli Top10 transformed with a kanamycin resistant plasmid (pET28A + or pEGFP-C2). The estimated D-values were 0,7 M for Sulfuric, 6,3% for a commercial P3 disinfectant, 25 minutes for steam sterilization at 121°C and 49 minutes for disinfection by UVC. A 20-minute treatment of bacteria cultures with a final concentration of 1–10% sodium hypochlorite was found to be ineffective in completely destroying a bacteria plasmid gene marker (coding for the pBR322 origin of replication). Residual DNA from HClO treated cells was 60%, while it decreased under 10% using the commercial disinfectant P3 diluted at 5%. As the degradation was uncomplete in both cases, to prevent the horizontal transfer of laboratory ARGs to environmental bacteria, disinfected liquid waste should not be released in sewage without additional plasmid destruction treatment.

Decontamination

Plasmid transfer

Antibiotic resistance gene

Antimicrobial resistance

Biosafety

horizontal gene transfer

Bacteria have developed various mechanisms of resistance to antibiotics, the first case of which was already described for penicillin in 1940 (Frère et al. 2016). Since 1985, the emergence of bacterial resistance has exceeded the discovery of new antibiotics. One explanation for the resistance rise is the overuse and misuse of antibiotics, which has contributed to the selection of bacteria expressing antibiotic resistance genes (ARGs). Thus, despite the implementation of legislation limiting the use of antibiotics in human and veterinary medicine, as well as in the food industries, in many countries (Bombaywala et al. 2021; Velazquez-Meza et al. 2022), the number of resistances in " superbugs" posing a serious threat to the success of traditional antibiotic therapy is steadily increasing (Poole 2002; Ventola 2015; Zaman et al. 2017; Bombaywala et al. 2021). Examples of these ‘superbugs’ include multi-drug resistant mycobacteria that cause tuberculosis (Ventola 2015) and the so-called ESKAPE pathogens, which include 6 leading pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. (Domingues et al. 2019). A recent survey using predictive statistical modeling the burden of Antimicrobial Resistance (AMR) for 23 pathogens and 88 pathogen-drug combinations in 204 countries indicated that they were responsible of more human deaths than AIDS or malaria in 2019 (Murray et al. 2022).

The horizontal transfer of plasmids or other mobile elements bearing ARGs (i.e. the ‘Resistome’) from commensal and environmental bacteria to clinical strains is the major mechanism of resistance acquisition (von Wintersdorf at al. 2016). Three mechanisms of horizontal gene transfer (HGT) have been described so far: the bacterial conjugation, the viral transduction of bacteria and the bacterial transformation. Although firstly considered as a minor mode of HGT, transformation of naturally competent bacteria in sewage and Waste Water Treatment Plants (WWTP) is increasingly considered an important transfer pathway (von Wintersdorf at al. 2016; Karkman et al. 2018).

At the interface between the environment and wastewater of anthropogenic origin, wastewater treatment plants are privileged places not only for the horizontal transfer of ARG between different bacterial taxa but also for the selection of antibiotic-resistant species by chemical pollutants (including antibiotics) present in wastewater (Rizzo et al. 2013; Berglund et al. 2015; Karkman at al. 2018; Che et al. 2019; Pazda et al. 2019). Therefore, the removal of antibiotics from hospital waste water is a key-objective to reduce their selective pressure in aquatic environments. A promising approach is the bioremediation by microalgae in symbiosis with bacteria (da Silva Rodrigues et al. 2021). Another key-challenge of WWTP is the successful extraction of ARGs and antibiotic resistant bacteria (ABR) during the water treatment. In this regard, while high-throughput PCR approaches and metagenomics data revealed the hosts or ARGs and their loss and gain over water treatments steps (Karkman et al. 2016; Martínez et al. 2017; Hultman et al. 2018; Che at al. 2019), other projects, dedicated to the assessment and/or development of neutralizing methods of ABR and ARG in WWTP effluents, highlighted many knowledge gaps to be filled before reaching an efficient methodology (Michael et al. 2013; Rizzo et al. 2013; Zheng et al. 2017; Noor et al. 2021; Uluseker et al. 2021; Nguyen et al. 2021; Yao at al. 2021; Wang et al. 2022).

In addition, the laboratory wastewater could represent an accidental release route of genetically modified microorganisms - harboring antibiotic resistance genes - in the environment. Indeed, in 2017 Bagutti and coworkers (Wichmann et al. 2017) mentioned that the detection of plasmids of genetically modified (GM) E. coli in laboratory wastewater (LWW) had already been reported in 1980. To prevent this risk, by the end of the 1990s regulations of contained used were implemented (Beeckman and Rüdelsheim, 2020) with the aim - among other goals - to make mandatory the decontamination and final destruction of biological waste. Despite this requirement, isolates of GM E. coli and synthetic plasmids were identified in the LWW of a Swiss university through the detection of universal plasmid primers (T7, M13, T3 and SP6) and sequencing (Wichmann et al. 2017).

Considering the potential contribution of research and production activities on the horizontal transfer of antibiotic resistance genes to wastewater bacterial populations, the first part of the present study was aimed at isolating antibiotic resistant bacteria from LLW and WWTP, and subsequently screening them for the presence of laboratory plasmid markers. The second aim of our project was the verification of the effectiveness of commonly used decontamination methods to degrade bacteria plasmids (DNA-dgr effect). Data gained in the present study permit to suggest decontamination approaches preventing the release of plasmids in wastewater from research laboratories.

Wastewater sampling

From March 2018 to April 2020, LWW of two research institutes was sampled at two sites: (1) the main sewage release pipes of laboratories from two building hosting molecular biology research units (referred as ‘MAS’ for ‘Medicine and Science’ samples) and (2) 14 laboratory sink traps (referred as ‘L’ for ‘Laboratories’ samples).

Sampling was also performed in two regional WWTPs, including hospital wastewater. The exact locations of the sampled LWW and WWTP are confidential (in agreement with site managers). The water samples (100 mL) were filtered through 0,22 µm Sterivex® filters (EMD Millipore, Germany) connected to a peristaltic pump (EMD Millipore, Germany) to concentrate bacteria. The filter was either placed in 10 mL of sterile water to resuspend bacteria before plating on selective media or directly plated on solid culture medium. For long-term use, bacteria cultures were conditioned as 60% glycerol stocks kept at − 80°C.

Detection of antimicrobial phenotypical resistance on selective media

Briefly, 0,1 mL of diluted bacterial suspension was plated on Luria-Bertani medium (LB 32 g/L Invitrogen, Yeast Extract 5g/L, BD and Peptone 6 g/L, BD), supplemented with one antibiotic used at the following concentrations: ampicillin, 100 µg.mL^− 1; kanamycin, 10 µg.mL^− 1; chloramphenicol, 20 µg.mL^− 1; nalidixic acid, 25 µg.mL^− 1 and spectinomycin, 100 µg.mL^− 1. Plates were incubated for 24 hours at 37°C. Then bacteria from morphologically different colonies were picked up and sub-cultured on the same media for further purification and identification.

Detection of the antibiotic resistance genes of wastewater isolates by PCR

The detection of the resistance genes frequently found in synthetic plasmids in bacteria isolates, was performed by colony-PCR using specific primers (listed in Table S1). PCR amplification and the subsequent detection of amplicons on gel electrophoresis were performed according to standard protocols (Ausubel, 2003).

Detection and sequencing of the gene of the replication origin pBR322 (ori gene)

Standard PCR conditions were used to detect the pBR322 origin of replication in E. coli plasmids. The primers used are detailed in Table S1.

PCR products were purified using the purification kit NucleoSpin® Gel and PCR Clean-up and sequenced using the Mix2Seq kit (Macherey-Nagel) by the Eurofins company (Belgium). Sequence identification and sequence comparison were performed using the BLAST program (Camacho et al. 2009) and the Clustal Omega multiple sequence alignment program available on the NCBI platform (Chojnacki et al. 2017). Before their final alignment, sequences were trimmed to the minimal length of analogous sequences.

Identification of bacteria genus / species through the sequencing of the 16S rRNA

Genomic DNA from bacteria cultures was purified using the kit NucleoSpin® ‘Genomic DNA from tissue’ (Macherey-Nagel). DNA concentrations were measured using a NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific). 16S rDNA was amplified by PCR using the primers listed in Table S1. The amplified product was purified using a PCR clean-up kit (NucleoSpin® Gel and PCR Clean-up), then sent for sequencing using the same primers and the Mix2Seq kit (Eurofins Genomics, Belgium). The bacteria were identified online on the NCBI platform, using the BLAST program (Camacho et al. 2009).

Testing the DNA degradation effect (DNA-dgr effect) of bacteria disinfection conditions

This part of the study (including growth assay described below) was performed using the bacteria strain E. coli One Shot™ Top10/DH10B (ThremoFisher, C404010/EC0113) containing the kanamycin resistant plasmid pET28A(+) (Novagen) or pEGFP-C2 (Clontech). For each treatment described below, samples of 50 µl of treated cultures were plated on agar supplemented with kanamycin 10 µg.mL-1) and incubated at 37° C for 48 h to check the effective death of the treated cells. No colonies were detected (data not shown).

Three chemicals and two physical treatments were tested for bacteria viability, as well as for integrity of DNA targets in the treated cells (sequences of the kanamycin resistance gene or sequences of the replication origin) using qPCR (see below). Viability was estimated through growth assays (see below).

Chemical disinfectants were (in final concentration): (1) NaClO (sodium hypochorite): 0,5–10% (v:v) of a liquid stock solution of NaClO 15° (Forever SA; 5% of Free Avalaible Chlorine or FAC, corresponding to 50 000 ppm); 0,1 − 0,5M NaOH, (2) Commercial P3 product: 1–10%, composed of a mix of Quaternary amine compounds (QACs; Didecyldimethylammonium chloride and Benzalkonium chloride) and others biocides (glyoxal, formaldehyde, glutaraldehyde and organic acids) and (3) acids: 0,1-2.5 M sulfuric acid and 0,1–2 M phosphoric acid. Treatments were performed using cultures presenting an final OD value at 600 nm (OD₆₀₀) of 0,65. Each chemical treatment lasted 20 minutes and was stopped by centrifugation (12 000 g for 30 seconds). Two physical treatments were tested: (1) steam at 121°C and (2) exposure to UVC (wavelength = 254 nm; high germicidal power). UVC exposure was carried out in a type II BSC (CleanAir, BioVangard). The 30W UV lamp had less than 500 hours of use and was positioned on the back wall of the cabinet, 53 cm above the cabinet work surface. The minimum intensity value of UVC irradiation was 30–225 mJ/cm², corresponding to exposures times between 5 and 30 minutes, at an intensity of 125 µW/cm² measured at 1 meter from the source. Since the samples were closer, the intensity - proportional to the squared distance - was multiplied by 3.6. For each exposure time, a rack of 4 tubes (2,5 ml capacity propylene tubes) containing 1 ml of culture of E. coli was positioned on the work surface, at the rear of the working surface of the BSC. A strip of aluminum was used to cover 1 tube serving as an unexposed control.

Following chemical or physical treatment, E. coli cultures were centrifuged at 12 000 g for 30 seconds and plasmid DNA was purified from the cell pellet using NucleoSpin® Plasmid EasyPure method (Macherey-Nagel) and diluted in a 20 µl in molecular biology grade water for PCR and qPCR.

Bacterial growth assays

The bacterial growth curves were performed using a bioscreen (Epoch2 Microplate Photospectrometer from BioTek). Bacterial cultures in exponential phase of growth were washed two times with PBS and were normalized to an OD₆₀₀ of 0,4 in a LB medium supplemented with the appropriate antibiotic. A growth-negative control was done using the growth medium devoid of the bacterial strain. A 200 µL aliquot of the normalized culture was transferred to a 96 wells plate and each condition was performed in technical triplicate (3 × 200 µL). The plate was incubated at 37 ◦C with shaking and the OD₆₀₀ of each well was measured every 10 min. Experiments were repeated at least three times to obtain biological triplicates. A second plate prepared in the same conditions was used to check the mortality of cells. Briefly, 100 µl of 5 samples taken at random from each series were centrifuged at 12,000 g for 30 seconds. The pellets were then resuspended in 100 µl of fresh LB medium before being plated (50 µl) on agar plates supplemented with 10 µg.mL − 1 kanamycin and incubated at 37°C for 48 h. No colonies were detected (data not shown)

Quantification of DNA degradation effect (DNA-dgr effect) of disinfection using quantitative PCR (qPCR)

Prior to qPCR, purified samples were diluted 10 and 50 times, to minimize possible interference of residual amount of chemical disinfectants on the PCR. Three microliters of the diluted samples were mixed with SyberGreen mix (FastStart universal SYBR Green Master Rox; Roche) and the appropriate primer sets (to amplify sequences of kanamycin resistance gene (KanR) or the gene of origin of replication pBR322 (or gene ori) and subjected to qPCR in a Light Cycler 96 (Roche). After checking that results were identical for ori and KanR, all experiments were only performed for gene ori. The DNA-dgr effect of each method was first checked on DNA standard, to check their possible interference with the extraction methods. After incubation at 98°C for 5 min, a total of 45 three-step cycles were performed as follows: 98°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec. Amplification cycles were followed by a final incubation at 72°C for 10 min. Three biological replicates and 3 technical replicates were performed for each qPCR.

The work was organized in two parts, in agreement with the double objective of the study i.e. the detection of plasmid gene markers in LWW and the identification of disinfection / decontamination methods of laboratory cultures achieving the degradation of antibiotic resistant plasmids.

Detection of the origin of replication of E. coli plasmids in LWW bacteria strains

To determine if laboratory strains are released in wastewater, we first collected water samples from exhaust drains of research facilities where the official methods of LLW disinfection are a 20-min incubation with sodium hypochlorite at a final concentration of 2,4 g.L^− 1 sodium hypochlorite (i.e., a 20-fold dilution of a stock solution with 5% of Free Available Chlorine or FAC). Bacteria isolates were selected using two criteria. The first selection criterion was the antibiotic resistance profile i.e., the capacity to grow on solid media containing antibiotics frequently used in research laboratories, namely: ampicillin, kanamycin, chloramphenicol, nalidixic acid and spectinomycin. The second criterion was the presence of the gene ori (origin of replication pBR322, found on many synthetic plasmids used in routine molecular biology technique) in the antibiotic resistant isolates. Among 16 antibiotic resistant isolates obtained from 8 LWW samples, 15 bacteria strains positive for the gene ori (Fig. 1) were selected for further analysis (samples L in Table S2). Applying the same approach with 36 bacteria strains isolated from research institute sewage exhaust ducts, 10 isolates positive for the ori gene were selected for further studies (samples MAS in Table S2). Finally, only 1 strain positive for the ori gene, out of 18 isolates from WWTP samples, was selected for further characterization (WWTP-1 in Table S2). The sequencing of the ori amplicons of most laboratory isolates (L1-6, L9-10, L12 and L15) displayed a 100% homology with that of the commercial synthetic plasmid used as positive control (pBR322 in Fig. 2).

The pBR322 origin of replication derives from the EcolE1 plasmid specific of Enterobacteriaceae and Pasteurellaceae (Ares-Arroyo et al. 2018, 2021). Therefore, without adding other origins of replication in these plasmids to broaden their host range, they could normally not replicate in other family bacteria (O’Neill and Bender 1988; Wu et al. 2010, Ares-Arroyo et al., 2018). However, most isolates positive for the ori gene obtained from wastewater samples analyzed in this study were not Enterobacteriaceae (Fig. 2). The presence of this typical marker of E. coli plasmids in non-Enterobacteriaceae bacteria found in wastewater suggests that it could have been acquired through horizontal transfer of laboratory plasmids that were released undestroyed in the sewage.

This is in line with not only the well described horizontal transfer of plasmid between bacteria, but also with its major role in ARGs dissemination (Sorensen et al. 2005 and Pinilla-Redondo et al. 2018). For instance, the colE1 plasmids, since they showed an increasing diversification of their antimicrobial resistance gene cargo - limited to bacteriocin before the antibiotic era - are considered key plasmids for the horizontal transfer of ARGs to other Enterobacteriaceae. (Ares-Arroyo et al. 2018; 2021 and Dolejska et al. 2018). Another example is the experimental demonstration of the transfer of IncHI1A plasmids not only within Enterobacteriaceae but also across other families such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae (Olesen et al. 2022). In line with a possible inter-family transfer of plasmids, in their recent publication Ares-Arroyo and coworkers (Ares-Arroyo et al. 2021) detected the non-conjugative ColE1 in non-enterobacteriaceae / non-Pasteurellaceae bacteria, namely: Pseudomonas, Vibrio, Alivibrio, Photobacterium and Aeromonas.

However, the detection of the pBR322 origin of replication in non-Enterobacteriaceae (non-Pasteurellaceae) would imply that this plasmid is stable and capable of replication in these bacteria strains. A possible explanation would be that this origin of replication is integrated in a broad host range plasmid possessing multiple origins of replication. This seems to be the case in certain strains of Aeromonas: beside a putative replication gene, they retain the ColE1 (pBR322) origin of replication itself (Ares-Arroyo et al. 2021).

Considering the natural competence of environmental bacteria, our data stress the necessity to avoid the accidental release of antimicrobial resistant genes in the sewage. This prompted us to investigate the effectiveness of bacteria disinfection methods routinely used in research facilities to degrade bacteria DNA (or DNA-dgr effect) in treated cells.

Testing the DNA degradation effect (DNA-dgr effect) of disinfection methods commonly used in the laboratory

As recently pointed out in reviews on this topic (Yoo 2018; Jones and Joshi 2021) the choice of disinfection methods should rely on in-depth knowledge of their mode of action in view of the increasing number of microorganisms resistant to chemical antimicrobial agents. The disinfection method must also be validated for each experimental situation, because - given the highly variable experimental conditions from one research laboratory to another - not all decontamination agents will be effective according to the protocol recommended by the normative texts (Uy et al. 2022). Moreover, showing the limited effect of very good biocides (such as glutaradehyde) to alter the integrity of cellular DNA, another recent study (Calderón-Franco et al. 2020) underlined the need to develop a strategy to overcome this shortcoming.

As a result, the present study focused on the identification of disinfection methods that would lyse bacteria and be accompanied by a degradation of DNA in treated cells, including plasmid-borne ARG.

We first investigated the impact of chemical disinfectants on the growth of the model bacterial strain E. coli TOP10 bearing the empty plasmid pET28A+ (biocidal effect). Using the same bacteria culture, the integrity of the plasmid pET28A + was assessed through the PCR detection of one of its gene markers: the pBR322 origin of replication. We tested the action of two frequently used biocides in research laboratories: sodium hypochlorite NaClO (stock solution with 5% of FAC) and a commercial disinfectant referred as P3 (composed of quaternary ammonium compounds or QACs, aldehydes and organic acid), both diluted to a final concentration of 0,5–10% (v:v). As shown in Fig. 3a and 3b, bacterial growth was inhibited at all tested concentrations of both biocides. By contrast, PCR data revealed that only P3 broke down the plasmid DNA (Fig. 3d), while no drastic reduction was observed in PCR products of sodium hypochlorite treated cultures (Fig. 3c). Assessment of DNA reduction using quantitative PCR confirmed that treatment with 10% NaClO led to a DNA maximal decrease of 40% (Fig. S4).

Using quantitative PCR, we quantified the DNA-dgr effect of disinfectants with the aim to determine their decimal reduction dose (D-value), reflecting the DNA resistance to a disinfection / decontamination procedure. Indeed, this value corresponds to the quantity of a chemical disinfectant, or the duration of a disinfection method, to decrease the plasmid DNA amount by 90%. The higher the D-value, the more ineffective the procedure. For chemical disinfectants tested over a given duration (20 minutes in the present study), the D-value is determined by the linear relationship between the residual quantity of DNA (expressed as log10 value) and the quantity of biocide used (in molar concentration or in %). The D-value of P3 for DNA degradation corresponds to a 6,3% final concentration of the stock solution. Determination of the D-value of NaClO for DNA degradation is not of interest under the disinfectant conditions applied in the present study, as it was found to be poorly effective.

Despite an efficient biocide effect of NaClO on bacteria cultures, incubation conditions applied in the present study failed to exert a complete DNA-dgr effect in dead cells. In agreement with our data, a the pre-treatment of the transforming plasmids with lower concentrations of FAC (between 0,5 and 5 mg.L^− 1) had no effect on the efficiency of transformation in vitro, suggesting that the treatment had no DNA-dgr effect (Öncü et al. 2011). However, considering the very low concentration used (compared to the conditions applied in the present study i.e. from 500 to 5000 mg.L^− 1 of FAC), one could not exclude that NaClO quantities used in their study were below the effective dose. Indeed, using higher quantities of FAC (i.e. 25 mg.L^− 1, which is still 20 times lower than the minimal quantity used in the present study), Zhang and coworkers (Zhang C et al. 2019a) observed the degradation of DNA of wastewater bacteria and a 1.7 log10 decrease in the transforming activity of their plasmids. In support to their observations, other reports indicated that chlorination is an effective method for destroying bacterial ARGs in wastewater treatment plants (Zheng et al. 2017 and Zhang M et al 2019b). However, the magnitude of the effect of the chlorine-releasing agent (CRA) depended on the antibiotic resistance gene, and the destruction of plasmid DNA was never complete (Zhang M et al 2019b; Liu and Hu, 2020). This is not entirely surprising considering that the first targets of CRAs (usually NaClO, ClO₂, HCLO and N-chloramines) in the process of disinfection are the membrane proteins of cells and organelles (Jones and Joshi 2021). Besides oxidizing proteins, CRAs also react with peptides and DNA itself, but proteins of cell membranes and organelles mobilizing a part of FAC, the resulting DNA-dgr effect on plasmid-resistant genes is reduced accordingly (matrix negative effect.) As a highly concentrated cell culture was used in the present study (corresponding to usual cultures of E. coli in research laboratories), it cannot be excluded that the poor DNA-dgr effect of NaClO results from the interference of cell protein (and other components of the culture medium) on this process, even at a high concentration of NaClO (10 times diluted stock solution, corresponding to 5 g.L^− 1of FAC). In line with this hypothesis, sodium hypochlorite is known to be sensitive to the presence of organic material in culture media: a higher concentration is required to achieve disinfection of culture media (2,5 g.L^− 1of FAC) and spillages of blood and body fluids (10 g.L^− 1of FAC), compared to surface disinfection (2,5 g.L^− 1of FAC) (Rutala &Weber 2015). Another oxidizing agent, peracetic acid (PAA) is mostly used for the surface disinfection of medical material by oxidizing macromolecules (Yoo 2018). Yet, in comparison with chlorine used at the same concentration (i.e. 25 mg.L^− 1), PAA is less powerful than chlorine to reduce the transforming activity of treated plasmids (Zhang C et al. 2019b) and is therefore not recommended for wastewater disinfection.

The commercial disinfectant P3 showed an efficient DNA-dgr effect in treated cells. The efficacy of this disinfectant cocktail results from the contribution of its components to destroy macromolecules (proteins and nucleic acids). For instance, QACs – acting as cationic surfactants - destabilize cell membranes (which results in cell lysis), while aldehydes present in the mix (formaldehyde, glutaraldehyde and glyoxal) also contribute to cell membrane disruption by chemically alkylating the amino (-NH₂) and sulfhydryl (-SH) groups of proteins, as well as the amino groups of nucleic acid bases (e.g. adenine) of DNA and RNA (Lin et al. 2020 and Jones and Joshi 2021). QACs also enhance the bactericidal effect of organic acids present in P3. Therefore, in disinfections procedures developed for laboratories, QACs are attractive biocides not only for the properties mentioned before, but also for their high tolerance towards the contaminating organic matter in cell culture. As a result, they efficiently open the way to aldehydes for DNA degradation, even in cultures with a high cell concentration (condition used in the present study).

Cultures of E. coli were also submitted to a disinfection treatment by acids, namely: 0,1 − 0,25 M sulfuric acid (Fig. S1a-c) or phosphoric acid (Fig. S1b-d) which were previously reported as efficient agents for DNA degradation in a former publication (Kochetkov 1972). Both treatments caused growth inhibition regardless of their respective concentrations (Fig.S1a-b), but sulfuric acid was more effective at degrading DNA, as seen in PCR results (Fig.S1c-d). Sodium hydroxide and hydrogen peroxide also exerted a strong inhibitory effect on bacteria growth (Fig S2a-b), but without perceptible effect on the plasmid DNA degradation (Fig. S2c-d) after a 20-minute treatment. D-values deduced from quantitative PCR data were respectively 0,69 M for sulfuric acid, 4.2 M for phosphoric acid and 3.2% for P3 (Fig. 4).

The high quantity of phosphoric acid (> 4M) is needed to reduce the DNA amount by only 1 log10. Therefore phosphoric acid is not suitable for DNA destruction in routine use, considering its high D-value. By contrast, the D-value of sulfuric acid determined in the present study (0,7 M) makes it a good disinfectant. However, the routine use of sulfuric acid as a disinfectant being hazardous to user health, as well as to the material integrity, it is also not recommended for routine disinfection.

Finally, we compared two physical methods of disinfection often used in laboratories: steam sterilization at 121°C to exposure to ultraviolet light (UVC). Both treatments led to DNA degradation in a time-dependent manner (Fig. S3a-b), confirmed using qualitative PCR. Sterilization at 121°C appeared a more rapid way to destroy plasmid DNA, as the D-values were determined as respectively being 25 minutes for steam sterilization at 121°C and 49 minutes for UVC irradiation (Fig. 4).

UVC are routinely used to decontaminate biosafety cabinets and hospital and healthcare settings (Scott at al. 2022), as well as to degrade DNA contaminating PCR workstations. Though UVC are the most germicidal amongst UV, it is surprising to see that UVC effects are rather slow to degrade DNA of bacteria cultures. Yet, recent reports of UV effect on plasmid and ARG integrity are converging to the same conclusion. For instance, apart from Zheng and coworkers’ data (Zheng et al. 2017), after UV irradiation of wastewater, there still are antibiotic resistant organisms and ARG in the effluent of a treatment plant (Guo and Kong 2019; Liu and Hu 2020), due to the matrix effect of liquid waste. This was also shown in attempts for food sterilizing using UV irradiation (Ballari and Martin 2013).

How to use the DNA-degradation D-values to design a disinfection method for laboratory liquid waste

As mentioned above, the D-value reflects the ineffectiveness of a DNA-degradation process in terms of biocide concentration or biocidal treatment time carried out under a given condition (concentration, sterilization temperature, exposure to UVC, etc.). When comparing disinfection approaches, the smaller the D-value, the more efficient the process. Using the D-value, it is possible to assess the Minimum Effective Concentration (MEC) defined as the lowest concentration of a chemical or product, used in a specified process, that achieves an activity claimed, expressed as log10 reduction of bacteria count. Many normative documents have been developed to test a wide variety of disinfectants activity by suspension and surface-carrier tests. Regarding bactericidal activity of disinfectants (European standard EN 14885:2018, in Tyski et al. 2022), MEC corresponds to a log reduction ≥ 4.0 in bacteria count.

MEC could also be assessed for the DNA-dgr effect of chemical disinfectants on the cell DNA. Under the conditions used in the present study, reaching this 4-log10 reduction of plasmid DNA in treated cells, would mean using a 20-minute incubation with a final concentration of 25.2% P3 or 2.8 M sulfuric acid (as deduced from results illustrated in Fig. 4). The 4-log10 reduction will also be obtained with steam sterilization performed during 100 min at 121°C or an UV irradiation performed for 3.3 h. Practically speaking, those conditions seem not manageable in routine, especially for chemicals. Using P3 (and mild acids such as PAA), the D-value could be decreased to a suitable one, using higher incubation time (60 min instead of 20 min, for example). Regarding steam sterilization, the D-value can also be decreased using a higher temperature (134°C). Those options to decrease D-values of DNA-dgr effect of disinfection methods will be the next step of our work, including also in vitro biological tests of the transforming capacity of treated samples.

Another alternative is taking advantage of synergies of combined disinfection methods. For instance, preliminary results (Fig. S4) showed that combining 10% sodium hypochlorite with a 30-minute UVC irradiation of a E. coli culture was more effective to degrade plasmid DNA (1.3-log10 decrease) than the sum of individual effects of NaClO (0,23-log10 decrease) and a 30-min UVC irradiation (0,74-log10 decrease). Although the statistical significance of this preliminary result should be confirmed, it is supported by similar synergies reported for the combination of UVC and chlorine to reduce the conjugative transfer (Lin et al. 2016) or to degrade ARGs (Liu and Hu 2020). The synergy could be explained by a better access of UV to the DNA following bacterial lysis by chlorine, combined with a reduced capacity of the DNA repair systems of the bacteria under double treatment.

Combining autoclaving with an oxidizing disinfectant such as ozone could also be an option. Indeed, using an approach resembling the method applied in the present paper, Lan and co-workers (Lan et al. 2021) detected only a limited decrease of ARGs in bacteria even after a 60-minute autoclaving, while they obtained a higher reduction of ARGs when autoclaving was followed by ozonation. Similarly, Zhang and coworkers (Zhang T. et al. 2019c), obtained a better degradation of the sulI antibiotic resistance gene using a treatment combining UV and chlorination, than by applying each approach separately.

Besides the advantages of combining two approaches, the extracellular organic matter (EOM) of cell culture being one of the major limiting agents of chemical disinfection, the design of disinfection procedures would improve significantly if one could establish a correlation between EOM quantities and another parameter indicative of the cell number in the culture, such as the optical density at 600 nm (for bacteria). The amount of protein in culture media could thus help to assess a maximal threshold value of cell culture OD allowing for the use of organic matter sensitive chemicals.

Considering the increasing number of evidences about the capacity of microorganisms to develop resistance to biocides (Russell 2002, 2003), which in turn could lead to increased resistance to antibiotics (Maillard 2018; Dopcea et al. 2020), methods combining chemical and physical disinfections seem to be the appropriate way to limit the emergence of resistance, as it will avoid using increased amounts of chemicals to degrade plasmid DNA. The resistance to QACs is well documented in Gram-positive and Gram-negative bacteria (Zou et al. 2004). It results from their rejection by non-specific efflux pumps also used to expel antibiotics from the cell. This might explain why exposing bacteria to a sublethal concentration of CAQs can lead to cross-resistance with other antibiotics like fluoroquinolones (Morrison et al., 2019). CAQs are also suspected to induce mutations leading to the development of resistance to antibiotic, as demonstrated by exposing E. coli to sublethal doses of benzalkonium chloride (BAC) or Didecyldimethylammonium chloride (DDAC) (Jia et al. 2022), which is especially alarming. As a result, two recommendations were recently issued regarding the use of chemical disinfectants. Firstly, biocides should only be used in their minimal inhibitory concentration (MIC) (Vijayakumar and Sandle, 2019). However, as seen in the present study, MIC doses of some disinfectants would not completely destroy plasmid DNA. The second recommendation is an urgent call to apply the current policy about antibiotic use to antiseptics / biocides, in order to preserve their biocidal benefits (Dopcea et al. 2020). Both recommendations stress the need to develop educational programs on the appropriate use of disinfectants in research laboratories, especially in low level containments (Biosafety level 1 and 2 laboratories) where the low risk of acquiring an infection in the workplace could lead to an underappreciation of the high risk of dissemination of ARGs – and therefore to contribute to the global antibiotic resistance crisis – due to accidental release of undestroyed plasmids in sewage.

Acknowledgments

The authors warmly thank Dr. Karim Zouai (Vésale hospital - Charleroi, Belgium), Salah Eddine Lali and Barbara Marchetti (Marie Curie hospital - Charleroi, Belgium), Pierre Bogaert, head of the molecular diagnostic laboratory (CHU UCL-Namur, Belgium) and Te-Din Daniel Huang, head of the clinical laboratory (CHU UCL-Namur, Belgium) for the fruitful scientific discussion on our data. We also thank Professor Bouchra Belkadi (Department of Biology of the University Mohammed V-Rabat, Morocco) for his support for Boutaina Habib's research stay at the University of Namur. We are particularly grateful to Professor Xavier De Bolle for allowing us to carry out this study in his laboratory and to benefit from the necessary equipment.

Funding

This work was supported by a grant from the continued education in Biosafety programmed developed by the prevention department (SerP) of the University of Namur. Boutaina Habib benefited from a PhD grant of the University Mohammed V (Rabat, Morocco), completed by a UNamur Grant for foreign students. Agnès Roba benefited from a PhD grant from the Belgian National Fund for Scientific Research.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Suzanne Loret designed the study, analyzed experimental data provided by Boutaina Habib and Pierre Romain and wrote the present paper. Boutaina Habib carried out the first part of the work (sampling, detection of antibiotic resistant isolates, sequencing of ori gene) under the supervision of Agnès Roba, who helped her to design primers and to use bioinformatic tools. Boutaina Habib also compiled the relevant literature to introduce the subject. She initiated the testing of disinfection methods under the tutoring of Angéline Reboul. Pierre Romain validated all results obtained by Boutaina Habib by quantitative PCR and confirmed the biocidal effects of disinfection treatment through growth assays.

Ethical approval – Not applicable

The present work did not involve human participants or animal experimentation.

Consent to participate – Not applicable

The research did not involve human subjects

Consent to Publish

All authors agreed with the content and gave explicit consent to submit the present data. They obtained consent from the responsible authorities at the institute/organization where the work has been carried out before the work is submitted.

All authors whose names appear on the submission

made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work;

drafted the work or revised it critically for important intellectual content;

approved the version to be published; and

agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Availability of data and material

Upon request authors are prepared to send relevant documentation or data to verify the validity of the results presented. This could be in the form of raw data, samples, records, etc. Sensitive information in the form of confidential or proprietary data is excluded.

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Prevention of horizontal transfer of laboratory plasmids to environmental bacteria: comparison of the effectiveness of a few disinfection approaches to degrade DNA.

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Abstract

Figures

Introduction

Materials And Methods

Wastewater sampling

Detection of antimicrobial phenotypical resistance on selective media

Detection of the antibiotic resistance genes of wastewater isolates by PCR

Detection and sequencing of the gene of the replication origin pBR322 (ori gene)

Identification of bacteria genus / species through the sequencing of the 16S rRNA

Testing the DNA degradation effect (DNA-dgr effect) of bacteria disinfection conditions

Bacterial growth assays

Quantification of DNA degradation effect (DNA-dgr effect) of disinfection using quantitative PCR (qPCR)

Results And Discussion

Detection of the origin of replication of E. coli plasmids in LWW bacteria strains

Testing the DNA degradation effect (DNA-dgr effect) of disinfection methods commonly used in the laboratory

How to use the DNA-degradation D-values to design a disinfection method for laboratory liquid waste

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