Description

1. Antibiotic resistance crisis: A Global Health burden

Antibiotic resistance emerges as a towering challenge in the contemporary global health landscape(Organization). The rising of antibiotic resistance is markedly accelerated due to abuse of antibiotic administration across human and veterinary medicine spheres. Today, WHO highlighted 12 bacterial families that represent the most significant danger to human health.(Organization, 2017, February 27). Recent data from 2019 highlighted the disastrous consequences of the antibiotic resistance, revealing the apex of global mortality linked to drug resistant bacteria predominantly stems from syndromes associated with lower respiratory infections (LRI+) and other thoracic infections("Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis," 2022)(Figure 1). Alternative strategies for combating drug resistant bacteria are urgently need for complementing traditional antimicrobial drugs, and overcome the antibiotic-resistance crisis.

2. Pseudomonas aeruginosa: A notorious opportunistic pathogen

Pseudomonas aeruginosa, a Gram-negative, aerobic, non-spore-forming rod-shaped bacterium, can cause a range of infections across hosts, regardless of immune status. Particularly, immunocompromised individuals like those with cystic fibrosis and diabetes are at greater risk. In healthcare settings, it's a primary agent for hospital-associated infections (HAIs) and is the second leading cause of ventilator-associated pneumonia in the US(Wu et al., 2015). Its ability to adhere to various surfaces and a vast arsenal of virulence factors enable effective colonization and infection, posing significant theraputic challenges(Moradali et al., 2017). The tendency of P. aeruginosa to acquire multi-drug resistance genetic elements amplifies the clinical challenge, highlighting the urgent need for innovative therapeutic interventions to mitigate the threats posed by this formidable pathogen(Penesyan et al., 2015).

3. Biofilm Bastions: a microbial stretegy for sscaping antibiotic eradication

Biofilms are architecturally complex assemblies of microbial entities that adhere to various surfaces, enveloped within a self-secreted Extracellular Matrix (ECM). This ECM is a conglomerate of diverse polymertic components, including exopolysaccharides (EPS), extracellular DNA (eDNA), RNA, proteins, and lipids, forming a robust physical barrier and chemical shield that safeguards the bacterial consortium from external threats, notably antibiotics(Costerton et al., 1999).

In P. aeruginosa , two critical exopolysaccharides, Pel and Psl, are well known to play important roles in biofilm formation. Pel, rich in glucose moieties, alongside Psl, which is mannose-dominant, orchestrate the initial irreversible attachment of bacterial cells to surfaces, contributing to the onset of biofilm formation(Colvin et al., 2011). This adhesion sets the stage for a community lifestyle, fostering microbial interactions, cell-to-cell communications and genetic material exchanges.

A remarkable feature within biofilms is the process of conjugation, which facilitates the horizontal transfer of genetic material among the microbial populations. This intra-community genetic exchange significantly amplifies the dissemination of antibiotic resistance genes, rendering the biofilm a reservoir for resistance propagation(Molin & Tolker-Nielsen, 2003).

The biofilm life cycle unfolds through a well-coordinated sequence of stages: the initial microbial attachment to surfaces, followed by biofilm maturation where the architecture diversifies and consolidates, and finally, the detachment of matured biofilm entities that may venture to colonize new niches (Lee & Yoon, 2017)(Figure 2). Each stage presents unique challenges and understanding these stages is crucial for designing strategies aimed at dismantling these microbial fortresses to restore antibiotic efficacy.

For the microbiological intricacies, the biofilm represents a formidable bastion that not only harbors but also fortifies the microbial community against the antibiotic onslaught, thereby demanding innovative therapeutic strategies to penetrate this microbial stronghold and abrogate the antibiotic resistance crisis.

4. C-di-GMP Signaling: The Biofilm Formation Master

Cyclic-di-GMP (c-di-GMP) emerges as a pivotal intracellular second messenger in P. aeruginosa and many Gram-negative species, orchestrating the complex 3D architecture development of biofilms by coordinating its effector proteins that govern motility, extracellular polymeric substance (EPS) synthesis, cell-to-cell communications etc (Ha & O'Toole, 2015; Hengge, 2009). The crescendo of c-di-GMP levels heralds a significant transition from reversible to irreversible cell attachment, setting the stage for mature biofilm formation. The molecular maestro, c-di-GMP, is synthesized and broken down primarily by the enzymatic duo of diguanylate cyclase (DGC) and phosphodiesterase (PDE), respectively.

During the biofilm maturation, a wspF-conducted surge in c-di-GMP levels amplifies the expression and activity of pel/psl genes, thereby augmenting the production of EPS, the building blocks of biofilm architecture(Andersen et al., 2021). A noteworthy mutation in the wspF gene leads to a ballet of increased cell aggregation and a visually discernible wrinkled colony morphology. wspF belongs to a gene ensemble encoding a signal transduction orchestra, a critical regulator that modulating the rhythmic movement of swimming-mediated chemotaxis in bacterial realms (D'Argenio et al., 2002).

Moreover, a fascinating encore is observed when the E. coli PDE yhjH is artificially induced in P. aeruginosa and P. putida , leading to a dramatic biofilm dispersal, and underscoring the pivotal role c-di-GMP and its regulatory ensemble play in modulating the biofilm dynamics (Andersen et al., 2021; Christensen et al., 2013). The nuanced understanding of c-di-GMP signaling pathway, akin to deciphering a complex musical score, is instrumental in devising innovative strategies to dismantle the resilient biofilm fortress, a step towards conquering the persistent P. aeruginosa infections.

5. Quorum Sensing: The Bacterial Parliament

Quorum sensing (QS) is a widely distributed mechanism employed by bacteria to regulate gene expression based on population density, coordinating virulence factors and biofilm formation, crucial for their pathogenicity and biofilms(Lee & Zhang, 2015; Wang et al., 2015). Among the myriad of strategies to thwart this microbial menace, the enzymatic disruption of QS, termed as quorum quenching, emerges as a promising avenue. Notably, enzymes aiiA and ytnP from other bacterial genera exhibit potent quorum quenching activity, thereby inhibiting the QS machinery of P. aeruginosa and subsequently attenuating its virulence(Djokic et al., 2022; Malešević et al., 2020).

aiiA specifically targets the N-acyl-l-homoserine lactone (AHL)-mediated QS pathways, leading to a substantial reduction in the expression of virulence factors and impeding biofilm development, thus showcasing its potential as a formidable tool in controlling bacterial infections(Anandan & Vittal, 2019; Pliuta et al., 2013). On a parallel note, a seminal study underscored the efficacy of Burkholderia cepacia-derived ytnP and Y2-aiiA lactonases in inhibiting the virulence of P. aeruginosa through quorum quenching(Malešević et al., 2020). Although the precise mechanism of action for ytnP remains elusive, its quorum quenching prowess hints at a role akin to aiiA , in modulating the QS system of P. aeruginosa .

The potential application of aiiA and ytnP in devising innovative therapeutic strategies to combat infections instigated by P. aeruginosa , particularly by targeting its QS system, holds promise for alleviating the global burden of antibiotic resistance.

6. Bacteriophage Resurgence: A Hopeful Horizon

The therapeutic challenge posed by P. aeruginosa infections is exacerbated by the existence of both the c-di-GMP signaling pathway and quorum sensing mechanisms, which to an extent, undermine the efficacy of antibiotic treatments(Opoku-Temeng & Sintim, 2017; Sikdar & Elias, 2022). Bacteriophage therapy, proposed early in the 20th century, has resurfaced as a viable alternative due to its specificity and ability to address cross-resistance issues.

A comparative analysis (Table 1) between bacteriophage therapy and conventional antibiotic therapy illustrates the merit of phage therapy. Unlike antibiotics which exhibit broad-spectrum activity, potentially disturbing non-target microbial entities, phages boast of high specificity targeting specific bacterial strains(Chegini et al., 2020). This specificity curtails the development of resistance and mitigates the impact on the host’s natural microflora(Debarbieux et al., 2010; Mboowa, 2023). Moreover, phages present a lower risk of cross-resistance owing to their unique mechanisms of action which diverge from those of antibiotics. Recent advances in phage-antibiotic combination therapies show promise, demonstrating synergistic effects in both lab and animal studies, hinting at a potential breakthrough in treating patients(Chegini et al., 2020).

Delving deeper into phage therapy, a pertinent discourse unfolds between virulent and temperate phage therapy, each with its unique set of merits and challenges as illustrated in Table 2. Virulent phages, operating through a lytic cycle, induce immediate bacterial lysis upon infection. This immediate lysis, while effective in rapidly reducing bacterial populations, may lead to the sudden release of endotoxins from the lysed bacterial cells, which could exacerbate inflammatory responses in the host. On the contrary, temperate phages operate through a lysogenic cycle, wherein they integrate their genome into the host's genome, delaying bacterial lysis. This delay in bacterial lysis potentially mitigates the risk of sudden endotoxin release, making temperate phage therapy a more controlled and less detrimental approach(Howard-Varona et al., 2017). Furthermore, temperate phages offer a unique platform for genetic engineering aimed at targeted biofilm disruption. Their ability to integrate into the host genome presents an opportunity to introduce genetic elements that can interfere with biofilm formation and antibiotic resistance mechanisms (Monteiro et al., 2019).

In summary, the choice of manipulating temperate phages over virulent phages hinges on the aim to:

• Minimize the risk of endotoxin release and associated inflammatory responses in the host.
• Utilize the lysogenic cycle as a vector for delivering genetic elements that can disrupt P. aeruginosa biofilm formation and antibiotic resistance mechanisms.
• Achieve a controlled and targeted therapeutic intervention against P. aeruginosa infections, which might offer a better safety profile and potentially lower the likelihood of resistance development compared to the abrupt lysis induced by virulent phages.

These considerations underscore the rationale behind opting for the modification of temperate phages in the quest for developing innovative strategies to tackle the challenge posed by P. aeruginosa infections.

7. Project Odyssey: Engineering Bacteriophages for Biofilm Disruption

To realize this objective, we engineered the temperate bacteriophage by integrating genes wspF , yhjH , aiiA , and ytnP into the pre-phage sequence pf4 of P. aeruginosa PAO1, each serving a distinct purpose towards biofilm management. Moreover, we amplified the expression of corresponding genes by utilizing high-copy-number plasmids in PAO1, serving as an indirect verification of gene functionality. For further validation, we constructed pf4 knockout strains, introducing the afore-mentioned plasmids to conduct rescue experiments. Through meticulous analysis, we observed the expression levels of the integrated genes and evaluated the subsequent impact on biofilm formation. Our endeavor aims at mitigating the adversities posed by P. aeruginosa infections by thwarting biofilm formation, strategically targeting the c-di-GMP pathway and quorum sensing mechanisms (Figure 3).

Reference

Anandan, K., & Vittal, R. R. (2019). Quorum quenching activity of aiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCL07 on AHL-mediated pathogenic phenotype in Pseudomonas aeruginosa. Microbial Pathogenesis, 132, 230-242.

Andersen, J. B., Kragh, K. N., Hultqvist, L. D., Rybtke, M., Nilsson, M., Jakobsen, T. H., Givskov, M., & Tolker-Nielsen, T. (2021). Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms. Antimicrobial Agents and Chemotherapy, 65(4), 10.1128/aac. 02431-02420.

Chegini, Z., Khoshbayan, A., Taati Moghadam, M., Farahani, I., Jazireian, P., & Shariati, A. (2020, Sep 30). Bacteriophage therapy against Pseudomonas aeruginosa biofilms: a review. Ann Clin Microbiol Antimicrob, 19(1), 45. https://doi.org/10.1186/s12941-020-00389-5

Christensen, L. D., van Gennip, M., Rybtke, M. T., Wu, H., Chiang, W.-C., Alhede, M., Høiby, N., Nielsen, T. E., Givskov, M., & Tolker-Nielsen, T. (2013). Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic di-GMP level in the bacteria. Infection and immunity, 81(8), 2705-2713.

Colvin, K. M., Gordon, V. D., Murakami, K., Borlee, B. R., Wozniak, D. J., Wong, G. C., & Parsek, M. R. (2011). The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS pathogens, 7(1), e1001264.

Costerton, J. W., Stewart, P. S., & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. science, 284(5418), 1318-1322.

D'Argenio, D. A., Calfee, M. W., Rainey, P. B., & Pesci, E. C. (2002, Dec). Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants. J Bacteriol, 184(23), 6481-6489. https://doi.org/10.1128/jb.184.23.6481-6489.2002

Debarbieux, L., Leduc, D., Maura, D., Morello, E., Criscuolo, A., Grossi, O., Balloy, V., & Touqui, L. (2010, Apr 1). Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis, 201(7), 1096-1104. https://doi.org/10.1086/651135

Djokic, L., Stankovic, N., Galic, I., Moric, I., Radakovic, N., Šegan, S., Pavic, A., & Senerovic, L. (2022). Novel quorum quenching ytnP lactonase from Bacillus paralicheniformis reduces Pseudomonas aeruginosa virulence and increases antibiotic efficacy in vivo. Frontiers in Microbiology, 13, 906312.

Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. (2022, Feb 12). Lancet, 399(10325), 629-655. https://doi.org/10.1016/s0140-6736(21)02724-0

Ha, D. G., & O'Toole, G. A. (2015, Apr). c-di-GMP and its Effects on Biofilm Formation and Dispersion: a Pseudomonas Aeruginosa Review. Microbiol Spectr, 3(2), Mb-0003-2014. https://doi.org/10.1128/microbiolspec.MB-0003-2014

Hengge, R. (2009, Apr). Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol, 7(4), 263-273. https://doi.org/10.1038/nrmicro2109

Howard-Varona, C., Hargreaves, K. R., Abedon, S. T., & Sullivan, M. B. (2017). Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME journal, 11(7), 1511-1520.

Lee, J., & Zhang, L. (2015, Jan). The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell, 6(1), 26-41. https://doi.org/10.1007/s13238-014-0100-x

Lee, K., & Yoon, S. S. (2017). Pseudomonas aeruginosa biofilm, a programmed bacterial life for fitness.

Malešević, M., Stanisavljević, N., Novović, K., Polović, N., Vasiljević, Z., Kojić, M., & Jovčić, B. (2020). Burkholderia cepacia ytnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity. Microbial Pathogenesis, 149, 104561.

Mboowa, G. (2023, 2023/10/03). Reviewing the journey to the clinical application of bacteriophages to treat multi-drug-resistant bacteria. BMC Infectious Diseases, 23(1), 654. https://doi.org/10.1186/s12879-023-08621-1

Molin, S., & Tolker-Nielsen, T. (2003). Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure. Current opinion in biotechnology, 14(3), 255-261.

Monteiro, R., Pires, D. P., Costa, A. R., & Azeredo, J. (2019, Apr). Phage Therapy: Going Temperate? Trends Microbiol, 27(4), 368-378. https://doi.org/10.1016/j.tim.2018.10.008

Moradali, M. F., Ghods, S., & Rehm, B. H. (2017). Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Frontiers in cellular and infection microbiology, 7, 39.

Opoku-Temeng, C., & Sintim, H. O. (2017). Targeting c-di-GMP Signaling, Biofilm Formation, and Bacterial Motility with Small Molecules. Methods Mol Biol, 1657, 419-430. https://doi.org/10.1007/978-1-4939-7240-1_31

Organization, W. H. Antibiotic resistance. https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance

Organization, W. H. (2017, February 27). WHO publishes list of bacteria for which new antibiotics are urgently needed. https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

Penesyan, A., Gillings, M., & Paulsen, I. T. (2015). Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities. Molecules, 20(4), 5286-5298.

Pliuta, V. A., Andreenko Iu, V., Kuznetsov, A. E., & Khmel, I. A. (2013). [Formation of the Pseudomonas aeruginosa PAO1 biofilms in the presence of hydrogen peroxide; the effect of the aiiA gene]. Mol Gen Mikrobiol Virusol(4), 10-14.

Sikdar, R., & Elias, M. H. (2022, Dec 21). Evidence for Complex Interplay between Quorum Sensing and Antibiotic Resistance in Pseudomonas aeruginosa. Microbiol Spectr, 10(6), e0126922. https://doi.org/10.1128/spectrum.01269-22

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Contribution

1. Temperate Phage Therapeutics: Genetic Engineering that Beyond Antibiotics

In the face of escalating antibiotic resistance, our project embarked on an expedition to unravel alternatives solutions for curbing bacterial infections. Through extensive literature review and consultations with field experts, we fully realized the burgeoning significance of phage therapy. Temperate phages furnish a unique scaffold for genetic engineering endeavors aimed at precise biofilm disruption. Their competency in integrating into the host genome opens the gateway to introduce genetic constructs capable of thwarting biofilm formation and antibiotic resistance machineries. Through this exploratory lens, our project not only elucidates the comparative merits of temperate phage therapy, but also propels the frontier of genetic engineering in combating antibiotic resistance, thereby contributing a novel perspective to the microbial warfare narrative.

2. Deciphering Biofilm Dynamics: The Game-Changing Potential of aiiA Gene Integration

Following an exhaustive review of the literature, we initially targeted four genes, wspF , yhjH , aiiA , and ytnP , for editing. Employing the introduction of high-copy-number plasmids for indirect validation, coupled with the elimination of the pf4 sequence for verification, we demonstrated the feasibility of all four genes as biofilm repressing elements. However, due to the high failure rate of homologous recombination, successful integration into the genome was achieved only for aiiA and yhjH . Through a series of molecular assays and biofilm formation experiments, we substantiated that the insertion of aiiA significantly curtails biofilm formation.

This investigative venture not only illuminates the potential of strategic gene editing but also accentuates the pivotal role of aiiA in biofilm dynamics. Our findings contribute to a broader understanding of microbial interactions and present a promising avenue for combating persistent biofilm-associated infections. Through meticulous genetic manipulation and experimental validation, our work epitomizes the confluence of synthetic biology and microbial ecology in devising innovative solutions for real-world microbial challenges.

3. Modeling Microbial Dynamics: Elucidating the Power of Gene Interference in Pseudomonas aeruginosa

To sum up, we developed three models. Firstly, we constructed a population growth model for P. aeruginosa using growth differential equations to calculate the concentrations of bacteria and viruses over time. This model elucidated the required burst size and infection rate for the virus to rapidly infect the entire colony. Subsequently, we conducted flux balancing analysis on a modified P. aeruginosa metabolic model by incorporating reactions related to the cyclic-di-GMP pathway. Simulation results showed that insertion of yhjH and wspF genes significantly inhibited c-di-GMP levels and biofilm formation. Finally, we constructed another differential equation model to investigate the impact of insertion of aiiiA and ytnP genes on quorum sensing in P. aeruginosa . By calculating critical points of the equations, our experiment demonstrated that the insertion of these genes significantly increased the activation threshold of quorum sensing and decreased its releasing threshold. Overall, our models support the notion that the insertion of our selected genes significantly impairs biofilm formation in P. aeruginosa , suggesting that mild bacteriophage therapy may serve as an effective treatment method.

Implementation

Overview

At present, antibiotic resistance in bacterial infections is very common, and Pseudomonas aeruginosa (P. aeruginosa ) infection is no exception. At present, the mechanism of drug resistance of P. aeruginosa mainly includes biofilm formation. Among them, there is a biofilm-forming pathway of P. aeruginosa population, its resistance increased by nearly 500-5000 times. Therefore, the formation of biofilm can be said to be an important reason for the resistance of P. aeruginosa . Our project aims to reduce resistance to P. aeruginosa by blocking the biofilm formation process. It can be seen from Figure 1 that there are two main ways to promote the biofilm formation of P. aeruginosa . The phage that we have modified contains molecules that can express these two pathways. The infection of P. aeruginosa by the phage can reduce the formation of biofilm and increase the sensitivity of P. aeruginosa to antibiotics.

Phage Engineering

Here, we engineered temperate phages with gene wspF , yhjH , aiiA , and ytnP integrated into P. aeruginosa PAO1's pf4 prophage sequence, each gene tailored for biofilm disruption. Employing high-copy-number plasmids in PAO1, we enhanced the expression of these genes, and via pf4 knockout strains, we executed rescue experiments to ascertain gene functionality (Figure 2). Our meticulous analysis delineated the expression dynamics of integrated genes and their impact on biofilm formation, underpinning a targeted therapeutic strategy against P. aeruginosa infections by intercepting the c-di-GMP pathway and QS mechanisms.

Following an exhaustive review of the literature, we initially targeted four genes, wspF , yhjH , aiiA , and ytnP , for editing. Employing the introduction of high-copy-number plasmids for indirect validation, coupled with the elimination of the pf4 sequence for verification, we demonstrated the feasibility of all four genes as biofilm repressing elements. However, due to the high failure rate of homologous recombination, successful integration into the genome was achieved only for aiiA and yhjH . Through a series of molecular assays and biofilm formation experiments, we substantiated that the insertion of aiiA significantly curtails biofilm formation (Figure 3). This investigative venture not only illuminates the potential of strategic gene editing but also accentuates the pivotal role of aiiA in biofilm dynamics. Our findings contribute to a broader understanding of microbial interactions and present a promising avenue for combating persistent biofilm-associated infections. Through meticulous genetic manipulation and experimental validation, our work epitomizes the confluence of synthetic biology and microbial ecology in devising innovative solutions for real-world microbial challenges.

Proof in modeling: Elucidating the Power of Gene Interference in Pseudomonas aeruginosa

To sum up, we developed three models. Firstly, we constructed a population growth model for P. aeruginosa using growth differential equations to calculate the concentrations of bacteria and viruses over time. This model elucidated the required burst size and infection rate for the virus to rapidly infect the entire colony. Subsequently, we conducted flux balancing analysis on a modified P. aeruginosa metabolic model by incorporating reactions related to the cyclic-di-GMP pathway. Simulation results showed that insertion of yhjH and wspF genes significantly inhibited c-di-GMP levels and biofilm formation. Finally, we constructed another differential equation model to investigate the impact of insertion of aiiiA and ytnP genes on quorum sensing in P. aeruginosa . By calculating critical points of the equations, our experiment demonstrated that the insertion of these genes significantly increased the activation threshold of quorum sensing and decreased its releasing threshold. Overall, our models support the notion that the insertion of our selected genes significantly impairs biofilm formation in P. aeruginosa , suggesting that mild bacteriophage therapy may serve as an effective treatment method (Figure 4).

Safety

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During the experiment, we realized that experimental safety is a very important thing. Therefore, we have always followed the following laboratory safety regulations to ensure the safety of each team member and the smooth progress of the experiment.

1. About our project

In our project design, we strictly adhere to all iGEM rules and policies. There are no organisms or activities in this project that exceed the iGEM whitelist or require prior permission from iGEM, and our data collection is unrelated to other humans, with almost no ethical or moral issues.

2. Biosafety rules

According to the infectivity of pathogenic microorganisms and the degree of harm to individuals or groups after infection, pathogenic microorganisms are divided into four categories:

• The first category of pathogenic microorganisms refers to those that can cause very serious diseases in humans or animals, as well as those that have not yet been discovered or have been declared extinct in our country.
• The second category of pathogenic microorganisms refers to those that can cause serious diseases in humans or animals and are relatively easy to spread directly or indirectly between humans, animals and humans, and animals and animals.
• The third category of pathogenic microorganisms refers to those that can cause human or animal diseases, but generally do not pose serious harm to humans, animals or the environment, have limited risk of transmission, rarely cause serious diseases after laboratory infection, and have effective treatment and prevention measures.
• The fourth category of pathogenic microorganisms refers to microorganisms that do not normally cause disease in humans or animals.

The first and second types of pathogenic microorganisms are collectively referred to as highly pathogenic microorganisms.

According to the biosafety protection level of the laboratory for pathogenic microorganisms, and in accordance with the provisions of the national standard for laboratory biosafety, the laboratory is divided into a level 1, a level 2 (which is divided into ordinary type and enhanced type), a level 3, and a level 4.

Experiments on pathogenic microorganisms shall be carried out in laboratories of corresponding grades. The low-grade pathogenic microorganism laboratory shall not engage in the pathogenic microorganism experiment activities that should be carried out in the high-grade pathogenic microorganism laboratory as stipulated in the national pathogenic microorganism catalogue.

The laboratory shall, on the basis of hazard assessment, develop standard operating procedures for specific experimental activities that have a greater impact on safety, including procedures for laboratory biosafety standard operation, testing research and experimental operation, use of relevant instruments and equipment, use of personal protective equipment, laboratory disinfection, disposal of hazardous waste, safe operation of sharp instruments and treatment of experimental emergencies.

The laboratory shall, in accordance with the relevant laws and administrative regulations on environmental protection and the provisions of the relevant departments under The State Council, dispose of waste water, waste gas and other wastes, and formulate corresponding environmental protection measures to prevent environmental pollution. The waste for designing experiments on pathogenic microorganisms must first be subjected to high temperature autoclave treatment. All waste must be sorted and temporarily stored, labeled and not discarded at will.

3. Bsl-2 requirement

• The door of the laboratory should have a window and can be locked, and the door lock and the opening direction of the door should not hinder the escape of indoor personnel.
• A sink should be provided and should be located near the exit of the laboratory.
• A clothing storage or hanging device should be set up at the entrance of the laboratory, and personal clothing and laboratory work clothes can be placed separately.
• The walls, ceilings and floors of the laboratory shall be easy to clean, impermeable to water and resistant to corrosion by chemicals and disinfectants. The ground should be smooth and non-slip, and carpet should not be laid.
• Laboratory cabinets and seats should be stable and their corners should be smooth.
• Laboratory cabinets and their placement should be easy to clean, and the experimental table should be waterproof, corrosion-resistant, heat-resistant and sturdy.
• The laboratory shall have enough space and cabinets for placing laboratory equipment and articles.
• Laboratory equipment, cabinets and articles should be placed reasonably according to the nature and process of the work to avoid mutual interference and cross-contamination, and should not interfere with escape and first aid.
• The laboratory may utilize natural ventilation. If mechanical ventilation is used, cross contamination should be avoided.
• If there are Windows that can be opened, install mosquito repellent screens.
• Unnecessary reflection and strong light should be avoided in the laboratory.
• If handling irritating or corrosive substances, eye washing facilities should be set up within 30 m, and emergency spray equipment should be set up if necessary.
• If handling toxic, irritating and radioactive volatile substances, appropriate negative pressure exhaust cabinets should be equipped on the basis of risk assessment.
• If highly toxic and radioactive substances are used, corresponding safety facilities, equipment and personal protective equipment shall be equipped, which shall comply with relevant national and local regulations and requirements.
• The door of the main entrance of the laboratory and the door of the laboratory where the biosafety cabinet is placed shall be automatically closed; The door at the main entrance of the laboratory shall have access control measures.
• There shall be conditions for the storage of spare materials outside the laboratory work area.
• Eye washing devices shall be provided in the laboratory work area.
• The laboratory or the building in which it is located shall be equipped with autoclaves or other appropriate disinfection and sterilization equipment based on a risk assessment.The laboratory or the building in which it is located shall be equipped with autoclaves or other appropriate disinfection and sterilization equipment based on a risk assessment.
• Biosafety cabinets shall be provided in laboratories where samples of pathogenic microorganisms are handled. The biosafety cabinet shall be installed and used according to the design requirements of the product. If the exhaust air of the biosafety cabinet is circulated indoors, the room should have ventilation conditions; If a biosafety cabinet is used that requires duct exhaust, it should be discharged through a duct independent of the other public ventilation systems in the building.
• There shall be a reliable supply of electricity. When necessary, important equipment (such as incubators, biosafety cabinets, refrigerators, etc.) should be equipped with backup power.

4. For bsl-2 lab, the configuration and operation of laboratory facilities and equipment shall follow the following guidelines:

(1) Use at all times a properly maintained biosafety cabinet, preferably a secondary biosafety cabinet, or other suitable personnel protection facilities or physical containment devices.

Experimental procedures to determine the possible formation of infectious aerosols or spillages include centrifuging, grinding, homogenizing, violent shaking or mixing, ultrasonic cracking, opening the container containing the infectious agent (the pressure inside the container may not be consistent with atmospheric pressure), animal nostril inoculation, and collection of infected tissue from animals or embryonic eggs.

When a high concentration or large volume source of infection is involved, if a centrifuge with a sealed rotor or a safety cover is used, if the rotor or safety cover is only opened in the biosafety cabinet, the centrifuge can be centrifuged in an open laboratory.

(2) When microorganisms must be handled outside the biosafety cabinet, facial protection measures (mirror, mask, mask, or other anti-splash device) should be taken to prevent infectious sources or other harmful substances from splashing or spilling on the surface.

(3) In the laboratory, special protective clothing, gown, smock or uniform must be used. When personnel go to non-laboratory areas, protective clothing must be left in the laboratory. Protective clothing can be handled in the laboratory or washed in the laundry room, but not taken home.

(4) Wear gloves when possible contact with potential sources of infection, contaminated surfaces or equipment. Two pairs of gloves are more appropriate. Gloves that are clearly contaminated should be disposed of and removed when the work on the source of infection is completed or the gloves are damaged. Disposable gloves do not need to be cleaned, cannot be reused, cannot be used to contact "clean" surfaces (keyboards, telephones, etc.), and should not be worn outside the laboratory. Have latex gloves with talcum powder. Wash your hands after removing gloves.

(5) The surface of the test bench should be waterproof, heat resistant, organic solvent resistant, acid and alkali resistant and durable in the workbench and other chemical substances for disinfection of facilities; There should be an eye irrigation device; If the laboratory has Windows facing the outside, window screens should be installed to prevent flies.

(6) When installing the biosafety cabinet, it is necessary to take into account the ventilation and exhaust of the room, which will not cause the biosafety cabinet to operate beyond normal parameters. The biosafety cabinet should be far away from the door, away from the window that can be opened, away from the walking area, and away from other equipment that may cause wind pressure confusion, to ensure that the airflow parameters of the biosafety cabinet are within the effective range.

5. Personal protective measures

(1) The laboratory director shall prohibit or restrict access to the laboratory while working on the source of infectious disease. In general, people who are susceptible or who will have serious consequences if infected are not allowed to enter the laboratory, for example, people with immune deficiencies or immunosuppression, who are at increased risk of infection. The laboratory director has the ultimate responsibility for assessing each situation and deciding who can work in the laboratory.

(2) Laboratory personnel receive appropriate immunizations or tests related to the source of the disease being treated or to be treated in the laboratory (e.g., hepatitis B immunization or TB skin tests); Where appropriate, basic serum samples appropriate for use by laboratory personnel and persons at risk are collected and stored, depending on the source of the disease being treated. Other serum samples should be collected regularly depending on the function of the disease source or facility being treated.

(3) Biosafety procedures shall be included in the standard operating procedures or biosafety manual specially developed by the laboratory Director for laboratory personnel. For persons with special risks, it is required to read and follow the work and procedures.

(4) The laboratory Director ensures that the laboratory and its supporting staff receive appropriate training, including possible risks associated with the work, necessary measures to prevent exposure, and procedures for exposure assessment. When changes in procedures are necessary, the personnel concerned must update their knowledge annually and receive additional training.

(5) In the presence of an exogenous source of disease, a biohazard sign shall be posted at the entrance to the laboratory, and the following information shall be displayed: the source of the disease, the biosafety level, the immunization requirements, the name of the researcher, the telephone number, the personal protective equipment that must be worn in the laboratory, and the procedures required for leaving the laboratory.

(6) Do not eat, smoke, wash glasses and make up in the work area. Food and daily necessities are not allowed in the work area. In the laboratory, people who wear hidden glasses also need to wear masks or face masks. Food should be stored in a dedicated cabinet or refrigerator outside the work area.

(7) Can not use mouth pipetting, only mechanical device pipetting; Develop safe use of sharp instruments. For contaminated sharp instruments, must always maintain a high degree of vigilance, including needles, syringes, slides, sampler, capillaries, scalpels; If you can use other tools, do not use sharp tools. When possible, use plastic instead of glass. Broken glassware can not be handled directly by hand, must be handled with other tools, such as brushes and dustpans, clips or tweezers. Containers containing contaminated needles, sharps and broken glass should be disinfected in accordance with regulations before they are dropped.

(8) All operating processes should be as careful as possible to avoid sputtering and aerosols; Disinfection should be carried out at least once a day, and tabletop disinfection should also be carried out in time when the living body spills. Spillage or accidental exposure to an apparent source of infection should be reported immediately to the laboratory director. Conduct appropriate medical evaluation, observation, treatment, and keep written records.

(9) All cultures, stores and other specified wastes shall be disinfected by feasible disinfection methods, such as autoclaving, prior to release. Materials transferred to the nearest laboratory for disinfection should be placed in durable, leak-proof containers, sealed and shipped out of the laboratory, and their packaging should comply with the relevant national regulations.