Induction of Biofilm Formation in Klebsiella pneumoniae ATCC 13884 by Several Drugs: The Possible Role of Quorum Sensing Modulation

Bacterial resistance is caused by several biochemical factors, the formation of biofilm being one of the main causes. This process is triggered by Quorum Sensing (QS), through the production of endogenous molecules, although other substances such as natural products can also do this. In this work, we aimed to determine whether some drugs are involved in the induction of biofilm formation in Klebsiella pneumoniae ATCC 13884, and thus, increase bacterial resistance. For this, the effect of 22 drugs on K. pneumoniae ATCC 13884 growth was determined at sub-plasmatic concentrations; the production of autoinducer lactones was established by HPLC and with a biosensor. The induction of biofilm formation was determined through crystal violet assay at 585 nm in a microplate reader and using urethral catheters. According to the in vitro assays, some drugs were found to induce biofilm formation in K. pneumoniae ATCC 13884. The effect of acetaminophen, hydrochlorothiazide, and progesterone stood out. The first drug caused several changes in the biochemistry of K. pneumoniae ATCC 13884 related to QS: high synthesis of N-hexanoyl-homoserine lactone, increasing bacterial populations by 27% and biofilm formation by 49%, and a more gentamicin resistant biofilm. Furthermore, it increased the colonization area of urethral catheters. Hydrochlorothiazide showed the biggest increase in the induction of biofilm formation of 51%, and progesterone displayed the greatest ability to provoke bacterial mass adherence but had no effects on K. pneumoniae ATCC 13884 bacterial population growth.

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Mutation of luxS Affects Biofilm Formation in Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.71.4.1972-1979.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 1972-1979 ◽

Cited By ~ 185

Author(s):

Justin Merritt ◽

Fengxia Qi ◽

Steven D. Goodman ◽

Maxwell H. Anderson ◽

Wenyuan Shi

Keyword(s):

Quorum Sensing ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Microscopic Analysis ◽

Homoserine Lactone ◽

Good Evidence ◽

Genome Project ◽

Bacterial Biofilm ◽

Wild Type

ABSTRACT Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html ). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

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Assessment of Antibiofilm Potencies of Nervonic and Oleic Acid against Acinetobacter baumannii Using In Vitro and Computational Approaches

Biomedicines ◽

10.3390/biomedicines9091133 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1133

Author(s):

Sagar Kiran Khadke ◽

Jin-Hyung Lee ◽

Yong-Guy Kim ◽

Vinit Raj ◽

Jintae Lee

Keyword(s):

Fatty Acids ◽

Oleic Acid ◽

Quorum Sensing ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Unsaturated Fatty Acids ◽

Homoserine Lactone ◽

Antimicrobial Treatment ◽

Dynamic Simulations

Acinetobacter baumannii is a nosocomial pathogen, and its biofilms are tolerant to desiccation, nutrient starvation, and antimicrobial treatment on biotic and abiotic surfaces, tissues, and medical devices. Biofilm formation by A. baumannii is triggered by a quorum sensing cascade, and we hypothesized that fatty acids might inhibit its biofilm formation by interfering with quorum sensing. Initially, we investigated the antibiofilm activities of 24 fatty acids against A. baumannii ATCC 17978 and two clinical isolates. Among these fatty acids, two unsaturated fatty acids, nervonic and oleic acid, at 20 μg/mL significantly inhibited A. baumannii biofilm formation without affecting its planktonic cell growth (MICs were >500 μg/mL) and markedly decreased the motility of A. baumannii but had no toxic effect on the nematode Caenorhabditis elegans. Interestingly, molecular dynamic simulations showed that both fatty acids bind to the quorum sensing acyl homoserine lactone synthase (AbaI), and decent conformational stabilities of interactions between the fatty acids and AbaI were exhibited. Our results demonstrate that nervonic and oleic acid inhibit biofilm formation by A. baumannii strains and may be used as lead molecules for the control of persistent A. baumannii infections.

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oxyR, a LysR-Type Regulator Involved in Klebsiella pneumoniae Mucosal and Abiotic Colonization

Infection and Immunity ◽

10.1128/iai.00837-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5449-5457 ◽

Cited By ~ 57

Author(s):

Claire Hennequin ◽

Christiane Forestier

Keyword(s):

Gastrointestinal Tract ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Defense Mechanisms ◽

In Vitro Assays ◽

Intestinal Epithelial ◽

Bacterial Transcription ◽

Host Interaction

ABSTRACT Colonization of the gastrointestinal tract is the first event in Klebsiella pneumoniae nosocomial infections, followed by colonization of the bladder or respiratory tract or entry into the bloodstream. To survive in the host, bacteria must harbor specific traits and overcome multiple stresses. OxyR is a conserved bacterial transcription factor with a key role both in the upregulation of defense mechanisms against oxidative stress and in pathogenesis by enhancing biofilm formation, fimbrial expression, and mucosal colonization. A homolog of oxyR was detected in silico in the K. pneumoniae sequenced genome and amplified from the LM21 wild-type strain. To determine the role of oxyR in K. pneumoniae host-interaction processes, an oxyR isogenic mutant was constructed, and its behavior was assessed. At concentrations lower than 107 ml−1, oxyR-deficient organisms were easily killed by micromolar concentrations of H2O2 and exhibited typical aerobic phenotypes. The oxyR mutant was impaired in biofilm formation and types 1 and 3 fimbrial gene expression. In addition, the oxyR mutant was unable to colonize the murine gastrointestinal tract, and in vitro assays showed that it was defective in adhesion to Int-407 and HT-29 intestinal epithelial cells. The behavior of the oxyR mutant was also determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment: deletion of oxyR resulted in higher sensitivity to bile and acid stresses but not to osmotic stress. These results show the pleiotropic role of oxyR in K. pneumoniae gastrointestinal colonization.

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Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase

Scientific Reports ◽

10.1038/srep16569 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 29

Author(s):

O. Lidor ◽

A. Al-Quntar ◽

E. C. Pesci ◽

D. Steinberg

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

In Silico ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Site Directed Mutagenesis ◽

Great Promise ◽

Acyl Homoserine Lactone ◽

Virulence Determinants

Abstract Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases.

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Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Marine Drugs ◽

10.3390/md19020074 ◽

2021 ◽

Vol 19 (2) ◽

pp. 74

Author(s):

Andrea Muras ◽

Ana Parga ◽

Celia Mayer ◽

Ana Otero

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Communication Systems ◽

Culture Medium ◽

Homoserine Lactone ◽

Bacterial Biofilm ◽

Screening Methods ◽

Time Saving ◽

Quorum Sensing Inhibition

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.

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Bacterial Communications in Implant Infections: A Target for an Intelligence War

The International Journal of Artificial Organs ◽

10.1177/039139880703000903 ◽

2007 ◽

Vol 30 (9) ◽

pp. 757-763 ◽

Cited By ~ 79

Author(s):

J.W. Costerton ◽

L. Montanaro ◽

C.r. Arciola

Keyword(s):

Quorum Sensing ◽

Bacterial Infections ◽

Bacterial Resistance ◽

Homoserine Lactone ◽

Bacterial Virulence ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Sensing Systems

The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called “quorum-sensing”. Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In gram-positive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

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Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.187.8.2870-2880.2005 ◽

2005 ◽

Vol 187 (8) ◽

pp. 2870-2880 ◽

Cited By ~ 102

Author(s):

Damien Balestrino ◽

Janus A. J. Haagensen ◽

Chantal Rich ◽

Christiane Forestier

Keyword(s):

Quorum Sensing ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Bacterial Species ◽

Microscopic Analysis ◽

Opportunistic Pathogen ◽

Signaling Molecules ◽

Pcr Analysis

ABSTRACT Quorum sensing is a process by which bacteria communicate by using secreted chemical signaling molecules called autoinducers. Many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density by this mechanism. In this study, the opportunistic pathogen Klebsiella pneumoniae was observed to secrete type 2 signaling molecules. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the K. pneumoniae genome. A V. harveyi bioassay showed the luxS functionality in K. pneumoniae and its ability to complement the luxS-negative phenotype of Escherichia coli DH5α. Autoinducer activity was detected in the supernatant, and maximum expression of specific messengers detected by quantitative reverse transcription-PCR analysis occurred during the late exponential phase. The highest levels of AI-2 were observed in minimal medium supplemented with glycerol. To determine the potential role of luxS in colonization processes, a K. pneumoniae luxS isogenic mutant was constructed and tested for its capacity to form biofilms in vitro on an abiotic surface and to colonize the intestinal tract in a murine model. No difference was observed in the level of intestinal colonization between the wild-type strain and the luxS mutant. Microscopic analysis of biofilm structures revealed that the luxS mutant was able to form a mature biofilm but with reduced capacities in the development of microcolonies, mostly in the early steps of biofilm formation. These data suggest that a LuxS-dependent signal plays a role in the early stages of biofilm formation by K. pneumoniae.

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Attenuation of Pseudomonas aeruginosa Quorum Sensing by Natural Products: Virtual Screening, Evaluation and Biomolecular Interactions

International Journal of Molecular Sciences ◽

10.3390/ijms21062190 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2190 ◽

Cited By ~ 3

Author(s):

Lin Zhong ◽

Vinothkannan Ravichandran ◽

Na Zhang ◽

Hailong Wang ◽

Xiaoying Bian ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Natural Products ◽

Quorum Sensing ◽

Biofilm Formation ◽

Bacterial Species ◽

In Vitro Assays ◽

Biomolecular Interactions ◽

Multi Drug Resistance ◽

In Silico Studies

Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these ‘super bugs’. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell–cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933±369 nM) and thermal shift assay further confirmed the biomolecular interactions. These results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.

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Interpopulation Signaling via N-Acyl-Homoserine Lactones among Bacteria in the Wheat Rhizosphere

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.11.1078 ◽

1998 ◽

Vol 11 (11) ◽

pp. 1078-1084 ◽

Cited By ~ 134

Author(s):

Elizabeth A. Pierson ◽

Derek W. Wood ◽

Jeffrey A. Cannon ◽

Francoise M. Blachere ◽

Leland S. Pierson

Keyword(s):

Gene Expression ◽

Antibiotic Production ◽

Homoserine Lactone ◽

In Vitro Assays ◽

Signal Molecule ◽

Wheat Roots ◽

Bacterial Populations ◽

Geographic Locations ◽

Wheat Rhizosphere

The biological control bacterium Pseudomonas aureofaciens 30-84 utilizes an N-acyl-homoserine lactone (AHL) signal molecule to control phenazine antibiotic production in the wheat rhizosphere (D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). In this study, we demonstrate that naturally co-existing, non-isogenic bacterial populations interact with strain 30-84 at the level of gene expression via the exchange of diffusible signals on wheat roots. Wheat plants from three geographic locations were used to generate a random library of 700 rhizobacterial isolates. Roughly 8% of the isolates from each location restored phenazine gene expression to an AHL-deficient strain of 30-84 in vitro. Five of these isolates were further tested for their ability to influence gene expression of an AHL-deficient reporter of strain 30-84 on wheat roots. All five, isolated from different geographic locations, restored phenazine gene expression by the reporter to wild-type levels. This suggests that in vitro assays can identify bacterial isolates with the potential to influence phenazine expression in strain 30-84 via AHLs on wheat roots. The occurrence of such strains in all fields sampled suggests that AHL-mediated communication is a common occurrence in the wheat rhizosphere.

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An In Vitro Coumarin-Antibiotic Combination Treatment of Pseudomonas aeruginosa Biofilms

Natural Product Communications ◽

10.1177/1934578x20987744 ◽

2021 ◽

Vol 16 (1) ◽

pp. 1934578X2098774

Author(s):

Jinpeng Zou ◽

Yang Liu ◽

Ruiwei Guo ◽

Yu Tang ◽

Zhengrong Shi ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Resistance ◽

Combination Treatment ◽

Crude Extract ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Antibacterial Properties ◽

Biofilm Growth ◽

Antibiotic Combination

The drug resistance of Pseudomonas aeruginosa is a worldwide problem due to its great threat to human health. A crude extract of Angelica dahurica has been proved to have antibacterial properties, which suggested that it may be able to inhibit the biofilm formation of P. aeruginosa; initial exploration had shown that the crude extract could inhibit the growth of P. aeruginosa effectively. After the adaptive dose of coumarin was confirmed to be a potential treatment for the bacteria’s drug resistance, “coumarin-antibiotic combination treatments” (3 coumarins—simple coumarin, imperatorin, and isoimperatorin—combined with 2 antibiotics—ampicillin and ceftazidime) were examined to determine their capability to inhibit P. aeruginosa. The final results showed that (1) coumarin with either ampicillin or ceftazidime significantly inhibited the biofilm formation of P. aeruginosa; (2) coumarin could directly destroy mature biofilms; and (3) the combination treatment can synergistically enhance the inhibition of biofilm formation, which could significantly reduce the usage of antibiotics and bacterial resistance. To sum up, a coumarin-antibiotic combination treatment may be a potential way to inhibit the biofilm growth of P. aeruginosa and provides a reference for antibiotic resistance treatment.

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