Application of bacterial growth regulator in regulation of clinical bacterial growth

Abstract

The invention belongs to the technical field of microbiology and medicine, and particularly relates to application of a bacterial growth regulator to regulation of clinical bacterial growth. The invention provides a novel bacterium growth regulator, namely sodium hydrosulfide (NaHS), and discloses a remarkable and complex bidirectional regulation effect of the sodium hydrosulfide on in-vitro growth of various clinically common gram-positive and gram-negative bacteria, and the sodium hydrosulfide can effectively inhibit growth of common gram-positive cocci and also can inhibit growth of common gram-negative cocci. The compound also shows a growth promoting effect on gram-negative enterobacter klebsiella pneumoniae and escherichia coli, and the promoting effect is proved through colony counting and dynamic growth curve monitoring. The discovery reveals the unique potential of the regulator as a bacterial growth regulation tool, and the regulator has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and pharmaceutical technology, specifically relating to the application of a bacterial growth regulator in regulating the growth of clinical bacteria. Background Technology

[0002] The continued spread of bacterial resistance has become a serious challenge to global public health, urgently requiring the search for new antimicrobial targets and strategies. Hydrogen sulfide (H2S), as an endogenous gaseous signaling molecule, plays an important role in various physiological and pathological processes. In recent years, research on H2S in the field of microbiology has also received increasing attention. Exogenous H2S donors, such as sodium hydrosulfide (NaHS), have been preliminarily reported to have growth-inhibiting effects on certain bacteria. However, systematic and comprehensive research data on the growth-regulating effects of NaHS as a bacterial growth regulator on a wide range of clinical isolates, especially its potential bidirectional regulatory effect (i.e., coexistence of inhibition and promotion) and its species specificity, are still lacking. Clarifying the specific effects of NaHS on these bacteria is crucial for assessing its value as a potential bacterial growth regulator (e.g., a candidate for novel antimicrobial agents or a specific microbial proliferation promoter) or a tool for studying the physiological characteristics of specific bacteria. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of a bacterial growth regulator in regulating the growth of clinical bacteria. Through a specific regulator, sodium hydrosulfide (NaHS), the growth of different types of bacteria can be effectively regulated to meet the needs of different fields such as medicine.

[0004] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0005] In a first aspect, the present invention provides the application of a bacterial growth regulator in regulating bacterial growth, wherein the bacterial growth regulator includes sodium hydrosulfide or a derivative thereof that can release hydrogen sulfide under physiological conditions.

[0006] The method of regulating bacterial growth includes at least one of the following (1)-(2):

[0007] (1) The regulator is used to inhibit bacterial growth, wherein the bacteria are at least one of Staphylococcus, Streptococcus, and Enterococcus;

[0008] (2) The regulator is used to promote bacterial growth, wherein the bacteria are Klebsiella pneumoniae and / or Escherichia coli.

[0009] This invention provides a novel bacterial growth regulator—sodium hydrosulfide (NaHS)—and reveals its significant and complex bidirectional regulatory effect on the in vitro growth of a variety of common clinical Gram-positive and Gram-negative enterobacteria. It not only effectively inhibits the growth of common Gram-positive cocci but also novelly exhibits a growth-promoting effect on common Gram-negative enterobacteria, including Klebsiella pneumoniae and Escherichia coli, which was confirmed by colony counting and dynamic growth curve monitoring. These findings reveal the unique potential of this regulator as a tool for bacterial growth control.

[0010] Preferably, the genus *Staphylococcus* includes at least one of *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus wari*, and *Staphylococcus hominis*.

[0011] Preferably, the Streptococcus genus includes at least one of Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus intermedius, and Streptococcus pyogenes.

[0012] Preferably, the Enterococcus genus includes Enterococcus faecalis and / or Enterococcus faecium.

[0013] Experimental studies have revealed that the growth regulator exhibits excellent broad-spectrum inhibitory properties, covering Staphylococcus, Streptococcus, and Enterococcus species. Furthermore, the growth regulator sodium hydrosulfide effectively promotes the growth of Klebsiella pneumoniae and Escherichia coli. Therefore, while existing technologies often target a single direction and struggle to address complex microbial structures where both pathogenic bacteria requiring inhibition and symbiotic bacteria requiring protection coexist, this invention, through the bidirectional regulatory effect of sodium hydrosulfide, can achieve both inhibition of specific pathogenic bacteria and promotion of key symbiotic bacteria within the same system. This provides a more flexible solution for complex scenarios such as intestinal microecological restoration and adjuvant treatment of infectious diseases.

[0014] More preferably, the streptococcus genus is at least one of Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus intermedius.

[0015] In a second aspect, the present invention provides a composition for regulating bacterial growth, the composition comprising sodium hydrosulfide or a derivative thereof capable of releasing hydrogen sulfide under physiological conditions, and further comprising pharmaceutically acceptable excipients.

[0016] Preferably, the composition is used to inhibit the growth of bacteria, wherein the bacteria are at least one of Staphylococcus, Streptococcus, and Enterococcus.

[0017] And / or, the composition is used to promote the growth of bacteria, said bacteria being Klebsiella pneumoniae and Escherichia coli.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] In existing technologies, bacterial growth regulators (such as antibiotics and bacteriostatic agents) mostly operate on a single mode of inhibition or promotion, and their selectivity for different bacterial species and strains is poor. For example, broad-spectrum antibiotics may indiscriminately inhibit a variety of bacteria, including beneficial bacteria. This invention, however, has discovered that sodium hydrosulfide (NaHS) can exhibit a complex bidirectional regulatory effect of "inhibition" or "promotion" on different bacterial species (Gram-positive cocci such as Staphylococcus spp., and Gram-negative bacteria such as Klebsiella pneumoniae). This achieves precise and differentiated intervention on the growth of specific bacteria, solving the problem of "single action and lack of selectivity" of traditional regulators. Attached Figure Description

[0020] Figure 1 These are representative agar plate photographs (disk diffusion method) showing the inhibitory effect of sodium hydrosulfide (NaHS) on the growth of different Staphylococcus strains described in this invention; including those against Staphylococcus aureus (…). Figure 1 A) and methicillin-resistant Staphylococcus aureus (MRSA) Figure 1 B) Staphylococcus epidermidis ( Figure 1 C) Staphylococcus warwick ( Figure 1 D) and Staphylococcus aureus ( Figure 1 The inhibitory effect of E).

[0021] Figure 2 These are representative agar plate photographs (disk diffusion method) showing the inhibitory effect of sodium hydrosulfide (NaHS) on the growth of different Streptococcus strains described in this invention; including those against Streptococcus pneumoniae (Streptococcus pneumoniae). Figure 2 A) Agalactococcus ( Figure 2 B) Intermediate Streptococcus ( Figure 2 C) and Streptococcus suis ( Figure 2 The inhibitory effect of D).

[0022] Figure 3 This is a graph showing the effect of the regulator sodium hydrosulfide (NaHS) described in this invention on the growth of Enterococcus strains (dilution plating method - modified plate pretreatment method); wherein... Figure 3 A and 3C represent the untreated NaHS control group. Figure 3 Groups B and 3D are NaHS treatment groups, demonstrating the effects of NaHS on Enterococcus faecalis ( Figure 3 A, Figure 3 B) and Enterococcus faecalis ( Figure 3 C Figure 3 D) Effects on growth.

[0023] Figure 4 The graph shows the effect of sodium hydrosulfide (NaHS), the regulator described in this invention, on the growth of Escherichia coli (dilution plating method - modified plate pretreatment method). Figure 4 A represents the untreated NaHS control group. Figure 4Group B, treated with NaHS, showed improved growth.

[0024] Figure 5 This is a graph showing the effect of the regulator sodium hydrosulfide (NaHS) described in this invention on the growth of Klebsiella pneumoniae (dilution plating method - modified plate pretreatment method); wherein... Figure 5 A represents the untreated NaHS control group. Figure 5 Group B, treated with NaHS, showed improved growth.

[0025] Figure 6 This is a summary plot of the inhibition zone diameter distribution of the regulator sodium hydrosulfide (NaHS) described in this invention against various common clinical Gram-positive cocci. This scatter plot summarizes the inhibition zone diameter (mm) produced by the regulator described in this invention against multiple clinical isolates of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus warwickii, Staphylococcus hominis, Streptococcus intermedia, and Streptococcus pneumoniae using the disk diffusion method.

[0026] Figure 7 This is a typical effect graph of sodium hydrosulfide (NaHS) as a regulator of the present invention on the growth curve of Escherichia coli. The graph compares the growth of a representative Escherichia coli (MZ1211-S) over time (h) under the conditions of adding NaHS (light blue curve, labeled "Escherichia coli + NaHS") and not adding NaHS (red curve, labeled "Escherichia coli"). Detailed Implementation

[0027] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0029] The modifier used in this invention is sodium hydrosulfide (NaHS·xH2O, analytical purity, purchased from Sigma-Aldrich, Cat. No. 161527), as a donor of hydrogen sulfide. Before use, it is freshly prepared to the required concentration with sterile deionized water or 0.9% physiological saline. The concentration of the NaHS stock solution used for the paper disc diffusion method is 400 mM, and the concentration of the NaHS working solution used for diluting the pretreatment of the coated plate is 100 mM.

[0030] The culture medium used for the strains described in the examples was MH medium and blood agar plate medium. Luria-Bertani (LB) broth was used for growth curve determination.

[0031] The strains described in this embodiment were all isolated from clinical samples, including blood, urine, sputum, and wound secretions. Clinical samples were spread on nutrient broth agar plates and incubated overnight at 37°C with 5% CO2. Single colonies were picked for microbial mass spectrometry identification. After identification, bacterial suspensions were prepared by streak subculturing and random selection of single colonies. The effect of NaHS on strain growth was investigated using the dilution plating method or disk diffusion method. Each bacterium tested in this invention was derived from approximately 2-10 samples. Figure 1-5 The results are for the test of a representative strain of each bacterium.

[0032] Example 1. Investigation into the inhibitory effect of the bacterial growth regulator on the growth of Staphylococcus spp.

[0033] This embodiment uses the paper disc diffusion method to investigate the inhibitory effect of the bacterial growth regulator sodium hydrosulfide on the growth of Staphylococcus spp., and the specific method is as follows:

[0034] (1) Preparation of strains: Collect clinically isolated Staphylococcus strains and prepare a 0.5 McFarland turbidity bacterial suspension;

[0035] (2) Paper diffusion experiment: Filter paper soaked with the regulator of the present invention (prepared from 400mM NaHS stock solution, each paper absorbs about 5μL) is attached to the surface of MH agar plates or blood agar plates inoculated with bacteria and incubated at 37°C for 18-24 hours.

[0036] (3) Results observation and analysis: The inhibitory effect of the regulator on the growth of the bacterial strain was investigated by measuring the diameter of the inhibition zone (mm);

[0037] 1. Staphylococcus aureus: The regulator inhibited both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains tested, forming visible inhibition zones. For example, the average inhibition zone diameter was approximately 19.5 mm for MSSA and approximately 18.2 mm for MRSA, indicating that the regulator was also effective against MRSA (see [link to relevant documentation]). Figure 1 A, 1B and Figure 6 ).

[0038] 2. Staphylococcus epidermidis: The regulator inhibited the tested Staphylococcus epidermidis, with an average inhibition zone of approximately 15 mm (see [link to relevant documentation]). Figure 1 C and Figure 6 ).

[0039] 3. Staphylococcus warwickii: The growth regulator inhibited the growth of the tested Staphylococcus warwickii, forming a visible inhibition zone with an average inhibition zone size of approximately 18.5 mm (see [link to relevant documentation]). Figure 1 D and Figure 6 ).

[0040] 4. Staphylococcus aureus: The regulator showed a significant inhibitory effect on the tested Staphylococcus aureus, forming a large inhibition zone with an average inhibition zone of approximately 34 mm (see [link to relevant documentation]). Figure 1 E and Figure 6 ).

[0041] Example 2. Investigation into the inhibitory effect of the bacterial growth regulator on the growth of Streptococcus spp.

[0042] The effects of the growth regulator (NaHS) on clinically isolated Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus intermedius were tested using the same test method as in Example 1.

[0043] See results Figure 2 A-2D and Figure 6 The growth regulator showed clear inhibition against the tested Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus intermedius, forming a clear inhibition zone. Figure 6 The average inhibition zone diameters of Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus intermedius were approximately 18 mm (2A), 22 mm (2B), and 17 mm (2C), respectively, while Streptococcus pyogenes showed relatively low sensitivity, with an average inhibition zone diameter of approximately 12 mm.

[0044] Example 3. Investigation into the inhibitory effect of the bacterial growth regulator on the growth of Enterococcus spp.

[0045] This embodiment investigated the inhibitory effect of sodium hydrosulfide, a bacterial growth regulator, on the growth of Enterococcus spp. using a plate dilution coating method with NaHS pretreatment. The specific method is as follows:

[0046] (1) Preparation of bacterial strains and working bacterial suspensions: Collect clinically isolated Enterococcus faecalis and Enterococcus faecium strains and prepare 0.5 McFarland turbidity bacterial suspensions respectively;

[0047] (2) Pretreatment of agar plates with conditioning agents: The treatment group was coated with 200 μL of 100 mM NaHS solution and allowed to dry; the control group was coated with physiological saline.

[0048] (3) Bacterial inoculation and culture: Take 20 μL of working bacterial suspension and inoculate it onto the pretreated plate. After incubation, count the colonies (CFU / plate).

[0049] (4) Results Analysis:

[0050] 1. Enterococcus faecalis: Compared with the control group without growth regulator treatment, the number of colonies on the plates was significantly reduced after growth regulator treatment, showing obvious growth inhibition (see...). Figure 3 A-3B).

[0051] 2. Enterococcus faecalis: Compared with the control group without growth regulator treatment, the number of colonies on the plates was significantly reduced after growth regulator treatment, showing obvious growth inhibition (see...). Figure 3 C-3D).

[0052] Example 4. Investigation into the promoting effect of the bacterial growth regulator on the growth of Enterobacteriaceae.

[0053] This embodiment tested the effect of the growth regulator (NaHS) on Escherichia coli and Klebsiella pneumoniae, and the specific method is as follows:

[0054] (1) Regulatory effect on Escherichia coli (using the plate dilution coating method with NaHS pretreatment):

[0055] Observation using the dilution-spreading method (same as in Example 3) showed that the growth regulator promoted the growth of the tested Escherichia coli. Compared with the control group, the number of colonies increased significantly after treatment with the growth regulator, demonstrating growth promotion (see [link to example]). Figure 4 AB).

[0056] (2) To further monitor and confirm the growth-promoting effect on Escherichia coli, growth curves of a representative Escherichia coli strain (MZ1211-S) were determined. The specific methods are as follows:

[0057] Single colonies were picked from fresh agar plates and suspended in Luria-Bertani (LB) broth. This bacterial suspension was then inoculated into 5 mL LB broth culture systems. Treatment groups were established with the regulator of this invention (10 mM NaHS) and a control group without the regulator, with three biological replicates for each treatment. Culture conditions were 35.5°C with shaking at 200 rpm. From the start of culture, 200 μL samples were aseptically collected every 4 hours (for a total of 28 hours), and their optical density (OD) was measured at 600 nm using a microplate reader. 600 Plot the average OD. 600 The growth curve of the value over time.

[0058] Growth curve results (see) Figure 7 ):like Figure 7 As shown, compared with the control group (Escherichia coli) without NaHS, the treatment group with NaHS (Escherichia coli + NaHS) showed a higher OD throughout the entire 28-hour culture period. 600The values ​​clearly demonstrate that the regulator described in this invention can significantly promote the growth of susceptible Escherichia coli strains, enabling them to reach and maintain higher cell densities more quickly.

[0059] (3) Regulation effect on Klebsiella pneumoniae (using the modified dilution plating method with NaHS pretreated plates): The same test method as in Example 3 was used.

[0060] Results analysis (see...) Figure 5 AB): Compared with the control group, the number of colonies of the tested Klebsiella pneumoniae strains increased significantly after treatment with the regulator, indicating growth promotion.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of a bacterial growth regulator in regulating bacterial growth, characterized in that, The bacterial growth regulators include sodium hydrosulfide or its derivatives that can release hydrogen sulfide under physiological conditions; The method of regulating bacterial growth includes at least one of the following (1)-(2): (1) The regulator is used to inhibit bacterial growth, wherein the bacteria are at least one of Staphylococcus, Streptococcus, and Enterococcus; (2) The regulator is used to promote bacterial growth, wherein the bacteria are Klebsiella pneumoniae and / or Escherichia coli.

2. The application as described in claim 1, characterized in that, The genus *Staphylococcus* includes at least one of *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus wari*, and *Staphylococcus hominis*.

3. The application as described in claim 1, characterized in that, The Streptococcus genus includes at least one of Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus intermedius, and Streptococcus pyogenes.

4. The application as described in claim 1, characterized in that, The Enterococcus genus includes Enterococcus faecalis and / or Enterococcus faecium.

5. A composition for regulating bacterial growth, characterized in that, The composition includes sodium hydrosulfide or a derivative thereof that can release hydrogen sulfide under physiological conditions, and also includes pharmaceutically acceptable excipients.

6. The composition according to claim 5, characterized in that, The composition is used to inhibit the growth of bacteria, wherein the bacteria are at least one of Staphylococcus, Streptococcus, and Enterococcus. And / or, the composition is used to promote the growth of bacteria, said bacteria being Klebsiella pneumoniae and / or Escherichia coli.

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