is one of the most common pathogens in nosocomial and community-acquired infections, which causes up to 10% of nosocomial infections.
1 K. pneumoniae
Predominant pathogens found in the European prevalence of infection in intensive care study.
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.
- Cadavid E
- Robledo S.M.
- Quiñones W
- Echeverri F.
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.
- Poulou A
- Voulgari E
- Vrioni G
- et al.
Outbreak caused by an ertapenem-resistant, CTX-M-15-producing Klebsiella pneumoniae sequence type 101 clone carrying an OmpK36 porin variant.
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.
- Mohamed SH
- Mohamed MSM
- Khalil MS
- Azmy M
- Mabrouk MI.
Combination of essential oil and ciprofloxacin to inhibit/eradicate biofilms in multidrug-resistant Klebsiella pneumoniae.
In the biofilm, bacteria stop growing to be adapted to hunger and hypoxia, reducing the action efficiency of antibiotics (antibiotics are effective against fast-growing bacteria).
- Piperaki ET
- Syrogiannopoulos GA
- Tzouvelekis LS
- Daikos GL.
Klebsiella pneumoniae: virulence, biofilm and antimicrobial resistance.
- Khodadadian R
- Rahdar HA
- Javadi A
- Safari M
- Khorshidi A.
Detection of VIM-1 and IMP-1 genes in Klebsiella pneumoniae and relationship with biofilm formation.
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,
- Guilhen C
- Miquel S
- Charbonnel N
- et al.
Colonization and immune modulation properties of klebsiella pneumoniae biofilm-dispersed cells.
making patients with biofilm bacteria have the characteristics of chronic infection or repeated infection.
- Durante-mangoni E
- Andini R
- Zampino R
Management of carbapenem-resistant enterobacteriaceae infections.
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.
Novel strategies for the prevention and treatment of biofilm related infections.
showed that medical devices coated with antibacterial agents effectively prevent biofilm colonization, but excessive use of the agents may cause microbial resistance. Studies
- Percival SL
- Slone W
- Linton S
- Okel T
- Corum L
- Thomas JG.
The antimicrobial efficacy of a silver alginate dressing against a broad spectrum of clinically relevant wound isolates.
- Eckhardt S
- Brunetto PS
- Gagnon J
- Priebe M
- Giese B
- Fromm KM.
Nanobio silver: its interactions with peptides and bacteria and its uses in medicine.
have shown that silver has antibacterial activity against planktonic bacteria and biofilm bacteria, but high concentrations of silver and other metals are toxic to human cells and tissues, so the use of metal ions is not safe. Other researchers
- Qian W
- Sun Z
- Wang T
- et al.
Antimicrobial activity of eugenol against carbapenem-resistant Klebsiella pneumonia and its effect on biofilms.
- Sah SK
- Rasool U
- Hemalatha S.
Andrographis paniculata extract inhibit growth, biofilm formation in multidrug resistant strains of Klebsiella pneumoniae.
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
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
(ESBLs) and carbapenemases, which acquire multiple drug resistance, may make infection challenging to treat.
- Mbelle NM
- Feldman C
- Sekyere JO
- Maningi NE
- Modipane L
- Essack SY.
Pathogenomics and Evolutionary epidemiology of multi -drug resistant clinical pneumoniae isolated from pretoria, South Africa.
As an essential factor in the mechanism of drug resistance, biofilm formation allows K. pneumoniae in vivo
to escape or resist the clearance of the host immune system and most antimicrobial agents, leading to chronic infection and failure of antibiotic treatment.
- Kaczmarek FM
- Dib-Hajj F
- Shang W
- Gootz TD.
High-level carbapenem resistance in a Klebsiella pneumoniaeclinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe, Antimicrob.
Updated functional classification of beta-lactamases.
Therefore, biofilm infection has become a significant problem in prevention and treatment. This study compared the biofilm production of the sensitive, ESBLs-producing, and carbapenem-resistant K. pneumoniae
on day 5. By measuring the effect of sodium hypochlorite on the 3 kinds of K. pneumoniae
biofilm, to some extent, the results show that sodium hypochlorite has inhibitory and clearance effect on the formation of K. pneumoniae
biofilms with different drug resistance, and the effect was enhanced with the increase of concentration in the range of bacteriostatic and bactericidal concentration.
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.
- Salles MM
- Oliveira Vde C
- Souza RF
- Silva CH
- Paranhos Hde F
Antimicrobial action of sodium hypochlorite and castor oil solutions for denture cleaning - in vitro evaluation.
- da Silva FC
- Kimpara ET
- Mancini MN
- Balducci I
- Jorge AO
- Koga-Ito CY.
Effectiveness of six different disinfectants on removing five microbial species and effects on the topographic characteristics of acrylic resin.
found that 0.25%, 0.5% and 1% sodium hypochlorite had the best anti-bacterial effect on tested microorganisms such as Candida, Streptococcus, Staphylococcus, Escherichia coli
. However, these studies only emphasized the killing effect of sodium hypochlorite on plankton; however, they did not evaluate its effect on biofilm formation. Some articles have reported the effect of sodium hypochlorite on S. aureus
biofilm and compared the activities of various disinfectants on S.s aureus
and Pseudomonas aeruginosa
biofilm, and observed that the activity of biofilm decreased significantly.
Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates.
- Gutierrez D
- Ruas-Madiedo P
- Martínez B
- Rodríguez A
- García P.
Effective removal of staphylococcal biofilms by the endolysin LysH5.
- Otter JA
- Vickery K
- Walker JT
- et al.
Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection.
Tiwari et al.
- Tiwari S
- Rajak S
- Mondal DP
- Biswas D.
Sodium hypochlorite is more effective than 70% ethanol against biofilms of clinical isolates of Staphylococcus aureus.
showed that sodium hypochlorite has a significant inhibitory effect on Staphylococcus
the biofilm formation of S. aureus
at working concentrations. Cai et al.
- Yanling C
- Hongyan L
- Xi W
- Wim C
- Dongmei D.
Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms.
found that sodium hypochlorite significantly reduced the activity of Enterococcus faecalis
biofilm, and the treatment effect was significantly improved with the increase of sodium hypochlorite concentration. However, the effect of sodium hypochlorite on the biofilm of K. pneumoniae
has not been reported. This study shows that sodium hypochlorite at concentrations of 200, 500, and 1,000 µg/mL inhibited the biofilm formation of 3 kinds of K. pneumoniae
with different resistance. Moreover, the inhibitory effect was improved with increased sodium hypochlorite concentration, consistent with the previous results.
Then we added sodium hypochlorite with 2,000 and 5,000 µg/mL concentrations on day 5 after biofilm formation, and we found that compared to the 2,000 µg/mL group, 5,000 µg/mL sodium hypochlorite had a more substantial clearance effect on biofilm of K. pneumoniae
with different resistance. Plutzer et al.
- Plutzer B
- Zilm P
- Ratnayake J
- Cathro P.
Comparative efficacy of endodontic medicaments & sodium hypochlorite against Enterococcus faecalis biofilms.
found that 4% sodium hypochlorite was the only agent that eliminated the biofilm of Enterococcus faecalis
in their experiment. Nair et al.
Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after “one-visit” endodontic treatment.
found that after dropping sodium hypochlorite on the biofilm, the in vitro
model often showed that the biofilm completely dissolved. Petridis and Clegg et al.
- Petridis X
- Busanello FH
- So MVR
- Dijkstra RJB
- Sharma PK
- van der Sluis LWM.
Factors affecting the chemical efficacy of 2% sodium hypochlorite against oral steady-state dual-species biofilms: Exposure time and volume application.
- Clegg MS
- Vertucci FJ
- Walker C
- Belanger M
- Britto LR
The effect of exposure to irrigant solutions on apical dentin biofilms in vitro.
found that biofilm is more easily removed by increasing the net number of reactive sodium hypochlorite molecules in contact with a specific biofilm surface area, and a similar increase in the net content of active sodium hypochlorite can be achieved by using a higher concentration of sodium hypochlorite (> 2%), which is consistent with the results of our study. Silva et al.
- Silva PM
- Acosta EJ
- Pinto LR
- et al.
Microscopical analysis of Candida albicans biofilms on heat-polymerised acrylic resin after chlorhexidine gluconate and sodium hypochlorite treatments.
and Gama et al.
- Gama MC
- Oliveira DG
- Silva PM
- Ordinola-Zapata R
- Duarte MH
- Porto VC.
Antifungal activity of 4% chlorhexidine and 2% sodium hypochlorite against Candida albicans biofilms.
evaluated the ability of a higher concentration of sodium hypochlorite to remove Candida albicans
biofilm after immersion, suggesting soaking times were 5 and 10 minutes. Badaró
- Badaró MM
- Bueno FL
- Makrakis LR
- et al.
Action of disinfectant solutions on adaptive capacity and virulence factors of the Candida spp. biofilms formed on acrylic resin.
recommended soaking time is 20 minutes, indicating that sodium hypochlorite is effective at a lower concentration by being in contact with the biofilm for more extended periods. Therefore, increasing the concentration or prolonging the soaking time removes K. pneumoniae
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.
The mechanism of sodium hypochlorite against bacterial biofilm may contain the following 2 points. First, the formation of most natural biofilms may start at a pH close to neutral.
- da Silva PM
- Acosta EJ
- Pinto Lde R
- et al.
Microscopical analysis ofCandida albicansbiofilms on heat-polymerised acrylic resin after chlorhexidine gluconate and sodium hypochlorite treatments.
The deviation of pH above or below 7 will significantly impact microbial metabolism and directly affect the properties of microorganisms and solid surfaces. It increases or decreases the electrostatic repulsion between the 2 entities, thereby interfering with the adhesion of microorganisms to the surface. Due to its alkaline pH (pH > 11), hypochlorite solution dissolves biofilm cells through increased electrostatic repulsion between bacterial cells and material surface.
Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes.
Secondly, Lineback proposed that sodium hypochlorite denatures proteins in the biofilm matrix and inhibit the function of major enzymes in bacterial cells, thus irreversibly killing bacterial cells in biofilms.
- Lineback CB
- Nkemngong CA
- Wu ST
- Li X
- 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.
Published online: January 01, 2022
Funding/support: This work was funded by Key Project of Natural Science Research of Anhui Universities of China ( KJ2020A0610 ), Young and Middle Aged Natural Science Foundation of Wannan Medical College (No. WK2021F26), Anhui Province's Natural Science Foundation (No. 2108085QH328), Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution Foundation of Wannan Medical College.
Conflicts of interest: We declare that we have no conflict of interest in this article.
Ethics approval and consent to participate: The isolation of the strain was part of the routine hospital laboratory procedure, without the need for informed consent and ethics committee approval. The study complied with the Declaration of Helsinki.
© 2022 The Author(s). Published by Elsevier Inc. on behalf of Association for Professionals in Infection Control and Epidemiology, Inc.