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Effect of sodium hypochlorite on biofilm of Klebsiella pneumoniae with different drug resistance

  • Chenlei Huang
    Footnotes
    Affiliations
    Department of Clinical Laboratory, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China
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  • Shaoneng Tao
    Affiliations
    Department of Nuclear Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China
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  • Jinlong Yuan
    Footnotes
    Affiliations
    Department of Neurosurgery, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China
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  • Xiaoning Li
    Correspondence
    Address correspondence to Xiaoning Li, MD, Department of Clinical Laboratory, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Zheshan West Road 2, Wuhu, Anhui 241000, China.
    Affiliations
    Department of Clinical Laboratory, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China
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  • Author Footnotes
    1 Contributed equally to the work and co-first authors.
Open AccessPublished:January 01, 2022DOI:https://doi.org/10.1016/j.ajic.2021.12.003

      Highlights

      • We studied the inhibitory and scavenging effects of sodium hypochlorite on biofilm of K. pneumonia with different drug resistance.
      • Crystal violet staining method combined with scanning confocal fluorescence microscopy was used to study the biofilm formation.
      • We provide a theoretical basis for the development of a reasonable strategy for the use of disinfectants to control nosocomial infections with K. pneumoniae.

      Abstract

      Background

      Biofilm formation is a major factor in the resistance mechanism of Klebsiella pneumoniae. This study aimed to evaluate the effects of sodium hypochlorite on the biofilm of K. pneumoniae with different drug resistance.

      Methods

      We collected 3 different types of K. pneumoniae respectively. The growth trend of biofilms of different drug-resistant K. pneumoniae was quantified by measuring the OD590 for 7 consecutive days using crystal violet staining. Scanning confocal fluorescence microscopy was used to observe biofilm morphology.

      Results

      After adding sodium hypochlorite, there were significant differences between the OD590 value of the 200, 500, and 1,000 µg/mL groups and the positive control group (all P < .05) on the fifth day. Concentrations of 2,000 and 5,000 µg/mL sodium hypochlorite were added after the biofilm had matured. In the 5,000 µg/mL sodium hypochlorite group, the OD590 of K. pneumoniae biofilm in the 3 groups decreased significantly compared with the blank control group (all P < .05).

      Conclusions

      Sodium hypochlorite inhibited and cleared the biofilm of K. pneumoniae with different drug resistance, and the effect was enhanced with the increase of concentration in the range of bacteriostatic and bactericidal concentration.

      Key words

      Introduction

      Klebsiella pneumoniae is one of the most common pathogens in nosocomial and community-acquired infections, which causes up to 10% of nosocomial infections.
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      Predominant pathogens found in the European prevalence of infection in intensive care study.
      K. pneumoniae exists in the respiratory tract and intestines of healthy people and animals and causes infection in various tissues and organs of the body, resulting in pneumonia, meningitis, liver abscess, urinary infection and sepsis.
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      Induction of biofilm formation in klebsiella pneumonia ATCC 13884 by several drugs:possible role of quorum sensing modulation.
      In recent years, with the wide application of antibiotics, the resistance of clinically isolated K. pneumoniae is becoming stronger and stronger. K. pneumoniae that produce extended-spectrum beta-lactamase (ESBLs) and carbapenem-resistant K. pneumoniae (CRKP) have been observed and introduced significant challenges to antibiosis.
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      Biofilm formation is a major factor in the resistance mechanism of K. pneumoniae. Biofilms are extracellular network-like aggregates of bacteria adhering to tissues, organs, and medical devices. They also form in a liquid culture medium. It is composed of polysaccharides, extracellular DNA and proteins encapsulated in bacterial aggregates. The antimicrobial resistance of K. pneumoniae in biofilm is 10-1,000 times higher than that in the planktonic state.
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      Some studies have shown that bacteria in the biofilm have stronger colonization ability and lower induction of immune system than planktonic bacteria, help bacteria escape the body's immune response and form an extracellular barrier to hinder the penetration of antibiotics,
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      making patients with biofilm bacteria have the characteristics of chronic infection or repeated infection.
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      Therefore, inhibiting or eliminating biofilm formation has increasingly become a significant problem in prevention and treatment.
      At present, anti-biofilm substances are mainly concentrated in antibiotics, metal ions and bioactive compounds. Chen et al.
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      showed that medical devices coated with antibacterial agents effectively prevent biofilm colonization, but excessive use of the agents may cause microbial resistance. Studies
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      showed that natural plant substances such as eugenol and Andrographis paniculata have significant inhibitory effects on K. pneumoniae biofilm. However, the natural compounds' structure and mechanism of action are not yet clear and may have unknown harmful effects on the human body. Disinfectants are a common means of nosocomial infection prevention and control that kill bacteria in vitro, thereby cutting off the transmission route to prevent pathogen infection and transmission. Sodium hypochlorite is a common disinfectant in hospitals; however, the effect on the biofilm of K. pneumoniae with different drug resistance is rarely studied. In addition, most of the current studies on K. pneumoniae biofilm only describe the inhibition or clearance effect. In contrast, we investigated the inhibition and clearance effect of sodium hypochlorite on the biofilm of K. pneumoniae by adding sodium hypochlorite on the first day of modeling and the day after the peak of biofilm formation. To the best of our knowledge, no such studies have been carried out. In addition, we further compared the effects of different concentrations of sodium hypochlorite on K. pneumoniae with different resistance, including sensitive, ESBLs-producing and CRKP subtypes, to better control the nosocomial infection and transmission of K. pneumoniae.

      Methods

      Materials

      K. pneumoniae of the sensitive (wild bacteria group), ESBLs, and CRKP groups were isolated from the inpatient specimens in several departments of Yiji Shan Hospital of Wannan Medical College from September 2019 to August 2020. There were 12 strains of each group. All were identified using a VITEK-2 Compact 60, a microbial identification instrument. The standard strain of K. pneumoniae was ATCC700603, and the quality control strain was Escherichia coli ATCC25922. All specimen collection processes were reviewed and approved by the hospital ethics committee. Sodium hypochlorite was purchased from Shandong Anjie Gaoke Disinfection Technology Co., Ltd. Beef paste broth and M-H broth were purchased from British OXOID Co., Ltd.

      Methods

      Preparation of bacteria suspension

      Thirty-six frozen bacteria were inoculated into a chocolate agar medium after rewarming, then incubated overnight at 37 °C. A single colony was picked and ground into 3 mL of sterile normal saline to formulate into a bacterial suspension with McFarland turbidity of 0.5 for standby (bacteria number was about 1.0 × 108 CFU/mL).

      Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of sodium hypochlorite

      MIC and MBC were measured in 96-well plates using the microdilution broth method. The sodium hypochlorite was prepared as a stock solution of 1,024 μg/mL for use and consecutively diluted to prepare working solutions of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg/mL. We also prepared sodium hypochlorite solutions at 1,000, 2,000, and 5,000 μg/mL. Thirty-six strains of bacteria formulated to a turbidity (optical density at 600 nm) of 0.5 of bacterial suspension, then diluted 100 times with sterile normal saline for use. We added 100 μL of bacteria suspension to each well of 96-well plates, and then 100 μL of sodium hypochlorite of various concentrations was added to each well, from low to high concentration. The positive control hole is bacterial suspension without sodium hypochlorite. The 96-well plates were incubated overnight at 37 °C statically, and the absorbance was measured using a microplate reader (Biotek Epoch2) at OD590.

      Detection of biofilms by crystal violet staining

      This method is improved based on Stepanovic et al.,
      • Stepanovic S
      • Vukovic D
      • Dakic I
      • Savic B
      • Svabic-Vlahovic M.
      A modified microtiter-plate test for quantification of staphylococcal biofilm formation.
      and the specific steps are as follows. The bacterial suspension prepared in the method part of Preparation of bacteria suspension was diluted 200 times so that the initial concentration was 5 × 105 CFU/mL, then incubated in Eppendorf tubes at 37 °C for 1-7 days statically. Next, we discarded the bacterial suspensions. The tubes were washed with sterile phosphate-buffered saline, fixed with methanol, stained with crystal violet, washed with sterile distilled water, and dissolved in 95% ethanol after the tubes were dried. Finally, we mixed the solution in the tubes and sucked 200 μL into each hole of the 96-well plates, respectively. The absorbance was measured for 7 consecutive days using a microplate reader at OD590.

      Effects of different concentrations of sodium hypochlorite on biofilm of K. pneumoniae with different resistance

      Biofilm inhibition test

      The MIC measured in the above experiments and the usual concentrations below the MIC (200, 500, and 1,000 μg/mL) were selected for the subsequent biofilm inhibition test. After diluting the bacterial suspension 10-fold, 50 μL were drawn into a sterile Eppendorf tube, mixed with 950 μL of beef extract broth, and culture at 37 °C until the biofilm is mature (the fifth day). The OD590 was measured using this method, and the biofilm inhibition rate was calculated (inhibition rate%=ODpositivecontrolOD590ODpositivecontrol×100%). The observation results of the broth to which the bacterial suspension has been added was taken as a positive control, and the broth without bacterial solution results were used as the blank control.

      Biofilm clearance test

      The MBC measured in the above experiments, and the usual concentrations above the MBC were selected for the subsequent biofilm clearance test. A total of 50 μL of the diluted bacterial suspension was drawn into a sterile Eppendorf tube, mixed with 950 μL of beef extract broth, and cultured in a 37 °C incubator for 5 days. Discard the bacterial suspension in the test tube, wash it twice with sterile phosphate-buffered saline, and add the broth medium with the corresponding concentration of sodium hypochlorite in each of the above test groups to Eppendorf tubes. The control group was added with an equal volume of broth medium without sodium hypochlorite. The suspension of each group was incubated at 37 °C for 24 hours. By repeatedly using the above method, biofilm production in tubes of each group was measured at 24 hours. Absorbance at 590 nm was determined, and the biofilm clearance rate was calculated (clearance rate% =ODcontrolOD590ODcontrol×100%). Observations of sensitive K. pneumoniae biofilms by confocal laser scanning fluorescence microscopy
      A small piece of autoclaved cover glass was added as the carrier into the Eppendorf tubes containing the bacteria solution and sodium hypochlorite broth. After the treatment described in 1.2.4.1 and 1.2.4.2, slides were created, and the images were obtained using a laser scanning confocal fluorescence microscope (Leica SP8) at an excitation wavelength of 576 nm.

      Statistical analysis

      We used SPSS17.0 for statistical analysis. Quantitative data were expressed as mean ± standard. The optical density of biofilm among groups was analyzed by one-way analysis of variance and LSD t-test. Differences were considered statistically significant when P < .05, and statistical charts were drawn using GraphPad Prism 6.0 software.

      Results

      Determination of MIC and MBC of sodium hypochlorite

      Sodium hypochlorite was set at various concentrations, and MIC and MBC were determined by visual observation of the liquid turbidity and determination of OD590. The MIC and MBC of the 36 strains were 1,000 μg/mL and 2,000 μg/mL, respectively.

      Detection of biofilms of K. pneumoniae with different resistance

      Thirty-six strains of 3 types of K. pneumoniae began to form biofilm on the first day after modeling and gradually increased over time, reached maturity on the fifth day, and began to decline after the fifth day. The OD590 of the 3 groups were measured from day 1 to day 7. The results measured on day 5 were as follows: sensitive group 0.648 ± 0.175, ESBLs group 0.685 ± 0.130 and CRKP group 0.637 ± 0.091 (Table 1). There were no significant differences among the 3 groups (P > .05). The growth trend of the biofilm of the 3 groups from day 1 to day 7 is shown in Figure 1.
      Table 1Biofilm yield of the 3 groups of bacteria on the 5th day
      Strain typeBiofilm yield on day 5F valueP value
      Sensitive group0.648 ± 0.1750.404.671
      ESBLs group0.685 ± 0.130
      CRKP group0.637 ± 0.091
      Note. Values are presented as the mean ± standard deviation.
      Fig 1
      Fig 1The process of biofilm formation in 3 groups of K. pneumonia. The biofilm of K. pneumonia began to form after modeling, and gradually increased with time, and reached maturity on the fifth day. ESBLs: extended spectrum β- Lactamases; CRKP: carbapenem resistant K. pneumonia. Values are presented as the mean ± standard deviation.

      Biofilm formation inhibition tests

      In the biofilm formation inhibition test, although biofilm production was gradually increased from days 1 to 5 after adding 200, 500, and 1,000 µg/mL of sodium hypochlorite, it is significantly reduced compared with the positive control (Fig 2). The biofilm formation process of the sensitive group after the addition of sodium hypochlorite is shown in Figure 2. On the 5th day, the OD590 of 200 μg/mL in the sensitive, ESBLs, and CRKP groups were 0.537 ± 0.078, 0.535 ± 0.069, 0.542 ± 0.0470, respectively; the OD590 of 500 and 1000 μg/mL in these 3 groups were 0.495 ± 0.078, 0.489 ± 0.056, 0.484 ± 0.063, and 0.433 ± 0.065, 0.432 ± 0.063, and 0.420 ± 0.073, respectively. According to the results of statistical comparison, the differences between positive control and 200, 500, and 1,000 μg/mL groups were statistically significant in these 3 types of K. pneumoniae (P < .05) (Fig 3). The inhibition percent reduction of 200, 500, and 1,000 µg/mL sodium hypochlorite on biofilm of K. pneumoniae in each group were 17.22%, 23.57%, 33.46% in the sensitive group, 16.12%, 23.21%, 32.40% in ESBLs group, and 14.30%, 24.1%, 34.35% in the CRKP group, respectively (Fig 4). Thus, sodium hypochlorite's inhibitory effect on the biofilm of 3 groups of K. pneumoniae increased with the concentration within the inhibitory concentration range.
      Fig 2
      Fig 2The process of biofilm formation of the sensitive group of K. pneumonia after the addition of sodium hypochlorite. After adding different concentrations of sodium hypochlorite, biofilm production of the sensitive group of bacteria increased gradually from the first day to the fifth day, but still significantly decreased compared with the positive control group.
      Fig 3
      Fig 3The inhibition test of different concentrations of sodium hypochlorite on the biofilms of sensitive group (3A), ESBLs group (3B) and CRKP group (3C) of K. pneumonia on the fifth day. The inhibition rate of the 3 groups has statistically significant differences at the concentrations of 200 µg/mL, 500 µg/mL and 1,000 µg/mL groups compared with the positive control group (P < .05). Among them, there was no significant difference between 200 µg/mL group and 500 µg/mL group in sensitive group and the ESBLs group (P > .05), while the difference between 200 µg/mL, 500 µg/mL and 1,000 µg/mL subgroup in the CRKP group was statistically significant (P < .05).
      Fig 4
      Fig 4The inhibitory percent reduction of different concentrations of sodium hypochlorite on 3 groups of K. pneumoniae biofilm. The inhibition percent reduction of sodium hypochlorite on biofilm was the highest in 1,000 µg/mL group.

      The statistical tests were one-way ANOVA and LSD-t test

      Biofilm clearance tests

      After 24 hours of incubation under the condition of 2,000 μg/mL sodium hypochlorite, the OD590 of the 3 groups were 0.608 ± 0.065 (sensitive group), 0.612 ± 0.066 (ESBLs group), and 0.619 ± 0.042 (CRKP group). The OD590 of the 3 groups after treatment of 5,000 μg/mL sodium hypochlorite was 0.561 ± 0.050 (sensitive group), 0.561 ± 0.044 (ESBLs group) and 0.563 ± 0.058 (CRKP group). According to the results of statistical comparison, there was no significant difference between the blank control group of the 3 groups of bacteria and the 2,000 µg/mL group (P > .05), while the difference between the blank control groups and the 5,000 µg/mL group were statistically significant (P < .05). In group CRKP, the difference between the 2,000 group and 5,000 µg/mL groups were also statistically significant (P < .05) (Fig 5). The clearance percent reduction of 2,000 and 5,000 µg/mL sodium hypochlorite on the biofilm of K. pneumoniae in each group were 5.07% and 12.26% in the sensitive group, 4.30% and 11.92% in the ESBLs group, and 3.38% and 12.14% in the CRKP group, respectively (Fig 6). After 24 hours of incubation by adding different concentrations of sodium hypochlorite, the clearance effect of sodium hypochlorite on biofilm of K. pneumoniae with different drug resistance increases with the increase of concentration within the range of bactericidal concentration.
      Fig 5
      Fig 5The clearance test of different concentrations of sodium hypochlorite on the biofilms of sensitive group (5A), ESBLs group (5B) and CRKP group (5C) of K. pneumonia. There was no significant difference between the blank control group of the 3 groups of bacteria and 2,000 µg/mL group (P > .05), while the difference between the blank control groups and 5,000 µg/mL group were statistically significant (P < .05). In group CRKP, the difference between the 2,000 µg/mL group and 5000µg/mL group were also statistically significant (P < .05).
      Fig 6
      Fig 6The clearance percent reduction of different concentrations of sodium hypochlorite on 3 groups of K. pneumoniae biofilm. The clearance percent reduction of sodium hypochlorite in 5,000 µg/mL group on biofilm was greater than in 2,000 µg/mL group.

      Observation of biofilms by laser scanning confocal fluorescence microscopy

      The inhibition and clearance of sensitive K. pneumoniae biofilms treated with various concentrations of sodium hypochlorite using laser scanning confocal fluorescence microscopy are illustrated in Figure 7. In the positive control group without sodium hypochlorite (Fig 7A), the extracellular polysaccharide matrix was connected into a network-like structure and agglomerates with each other. In contrast, the formation of extracellular polysaccharide matrix reduced after adding 200 (Fig 7B), 500 (Fig 7C), and 1,000 μg/mL (Fig 7D) sodium hypochlorite and dispersed with expanded pores. The formation of network-like structures was also reduced, leaving only a small number of scattered colonies. Figure 7G shows the biofilm clearance on day 5 observed after the addition of equal volumes of antibiotic-free broth medium (the blank control group). The extracellular polysaccharide matrix adheres and aggregate to form a grid-like structure. However, the extracellular polysaccharide matrix of the biofilm was decreased qualitatively, and the network structure was sparse and dispersed after adding 2,000 (Fig 7E) and 5,000 μg/mL sodium hypochlorite (Fig 7F).
      Fig 7
      Fig 7Images of biofilms in sensitive K. pneumoniae using laser scanning confocal fluorescence microscopy (40 ×). The inhibitory effect on biofilms in the positive control group(7A), 200μg/mL(7B), 500 μg/mL(7C), and 1,000 μg/mL(7D). The clearance effect on biofilms in the 2,000 μg/mL(7E), 5,000 μg/mL(7F) sodium hypochlorite and blank control groups (7G).

      Discussion

      In recent years, K. pneumoniae has become one of the primary pathogens causing nosocomial infections, causing high morbidity and mortality. K. pneumoniae carries a variety of antimicrobial resistance genes, including extended-spectrum β-lactamases (ESBLs) and carbapenemases, which acquire multiple drug resistance, may make infection challenging to treat.
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      In this study, we evaluated its inhibitory effect on the biofilm formation of K. pneumoniae by adding 200, 500, and 1,000 µg/mL sodium hypochlorite at the beginning of the experiment. We found that the biofilm yield of the 3 groups increased gradually from day 1 to day 5 after adding different concentrations of sodium hypochlorite; however, it decreased considerably compared with the positive control group, indicating biofilm formation ability was weakened after adding sodium hypochlorite. Previous studies have demonstrated the effect of sodium hypochlorite on bacterial growth. Salles and da Silva et al.
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      To determine whether the OD590 reflects the biofilm formation of K. pneumoniae, we observed the bacteria adhered to the slide with laser scanning confocal fluorescence microscope. In the positive control group without adding sodium hypochlorite, biofilms connected into a network and agglomerated with each other, while the biofilms gradually reduced after adding different concentrations of sodium hypochlorite, leaving only a small amount of scattered colonies. These findings agree with the results of absorbance measurements. From another point of view, it also suggests that sodium hypochlorite inhibits, scavenges the formation of K. pneumoniae biofilm, and shows that the decrease of OD590 reflected the decrease of biofilm quality.
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      • Teska PJ
      • Oliver HF.
      Hydrogen peroxide and sodium hypochlorite disinfectants are more effective against Staphylococcus aureus and Pseudomonas aeruginosa biofilms than quaternary ammonium compounds.
      However, there is insufficient evidence to explain how specific components attack biofilm integrity.
      This study has a few limitations. The microbroth dilution method was made by mixing a disinfectant with a nutrient broth to prepare a broth culture medium containing various disinfectant concentrations to cultivate bacterial suspension. This method has good repeatability and simply and interprets the results. However, for sodium hypochlorite, nutritional broth consumes substantial amounts of available chlorine; therefore, the accuracy of this method merits additional discussion. As a follow-up to this study, we will increase the types and quantities of samples to detect the anti-disinfectant gene of K. pneumoniae, to reveal further the mechanism of disinfectant resistance, help to formulate a reasonable strategy for the use of disinfectants and control the infection and transmission of K. pneumoniae in the hospital.

      Conclusion

      Sodium hypochlorite has inhibitory and clearance effects on the formation of biofilm of K. pneumoniae with different antibiotic resistance (sensitive, ESBLs and CRKP), and this effect was enhanced with the increase of concentration within the range of bacteriostatic and bactericidal concentration.

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