The In Vitro Ability of Klebsiella pneumoniae to Form Biofilm and the Potential of Various Compounds to Eradicate it from Urinary Catheters

Urinary infections related to the presence of bacterial biofilm on catheters are responsible for loss of patients’ health and, due to their high frequency of occurrence, generate a significant economic burden for hospitals. Klebsiella pneumoniae is a pathogen frequently isolated from this type of infection. In this study, using a cohesive set of techniques performed under stationary and flow conditions, we assessed the ability of 120 K. pneumoniae strains to form biofilm on various surfaces, including catheters, and evaluated the usefulness of clinically applied and experimental compounds to remove biofilm. The results of our study indicate the high impact of intraspecies variability with respect to K. pneumoniae biofilm formation and its susceptibility to antimicrobials and revealed the crucial role of mechanical flushing out of the biofilm from the catheter’s surface with use of locally active antimicrobials. Therefore, our work, although of in vitro character, may be considered an important step in the direction of efficient reduction of K. pneumoniae biofilm-related hospital infections associated with the presence of urine catheters.

Download Full-text

New Adapted In Vitro Technology to Evaluate Biofilm Formation and Antibiotic Activity Using Live Imaging under Flow Conditions

Diagnostics ◽

10.3390/diagnostics11101746 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1746

Author(s):

Cassandra Pouget ◽

Catherine Dunyach-Remy ◽

Alix Pantel ◽

Sophie Schuldiner ◽

Albert Sotto ◽

...

Keyword(s):

Biofilm Formation ◽

Cell Imaging ◽

Chronic Wounds ◽

Chronic Wound ◽

Live Imaging ◽

Bacterial Biofilm ◽

Flow Conditions ◽

Promising Tool ◽

Reference Strains

The polymicrobial nature of biofilms and bacterial interactions inside chronic wounds are keys for the understanding of bacterial cooperation. The aim of this present study was to develop a technique to study and visualize biofilm in live imaging under flow conditions (Bioflux™ 200, Fluxion Biosciences). The BiofluxTM system was adapted using an in vitro chronic wound-like medium (CWM) that mimics the environment encountered in ulcers. Two reference strains of Staphylococcus aureus (Newman) and Pseudomonas aeruginosa (PAO1) were injected in the BiofluxTM during 24 h to 72 h in mono and coculture (ratio 1:1, bacteria added simultaneously) in the CWM vs. a control medium (BHI). The quantification of biofilm formation at each time was evaluated by inverted microscopy. After 72 h, different antibiotics (ceftazidime, imipenem, linezolid, oxacillin and vancomycin) at 1x MIC, 10x MIC and 100x MIC were administrated to the system after an automatic increase of the flow that mimicked a debridement of the wound surface. Biofilm studies highlighted that the two species, alone or associated, constituted a faster and thicker biofilm in the CWM compared to the BHI medium. The effect of antibiotics on mature or “debrided” biofilm indicated that some of the most clinically used antibiotic such as vancomycin or imipenem were not able to disrupt and reduce the biofilm biomass. The use of a life cell imaging with an in vitro CWM represents a promising tool to study bacterial biofilm and investigate microbial cooperation in a chronic wound context.

Download Full-text

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.

Download Full-text

Identification of a Conserved Chromosomal Region Encoding Klebsiella pneumoniae Type 1 and Type 3 Fimbriae and Assessment of the Role of Fimbriae in Pathogenicity

Infection and Immunity ◽

10.1128/iai.00585-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 5016-5024 ◽

Cited By ~ 59

Author(s):

Carsten Struve ◽

Martin Bojer ◽

Karen Angeliki Krogfelt

Keyword(s):

Klebsiella Pneumoniae ◽

Gene Cluster ◽

Virulence Factor ◽

Biofilm Formation ◽

Regulatory Gene ◽

Infection Model ◽

Type 3

ABSTRACT Type 3 fimbriae are expressed by most clinical Klebsiella pneumoniae isolates and mediate adhesion to host structures in vitro. However, the role of type 3 fimbriae in K. pneumoniae virulence has not been evaluated by use of in vivo infection models. In this study, the type 3 fimbrial gene cluster (mrk) of the clinical isolate C3091 is described in detail. The mrk gene cluster was revealed to be localized in close proximity to the type 1 fimbrial gene cluster. Thus, a 20.4-kb fimbria-encoding region was identified and found to be highly conserved among different K. pneumoniae isolates. Interestingly, a homologue to PecS, known as a global regulator of virulence in Erwinia chrysanthemi, was identified in the fimbria-encoding region. Comparison to the previously characterized plasmid encoded mrk gene cluster revealed significant differences, and it is established here that the putative regulatory gene mrkE is not a part of the chromosomally encoded type 3 fimbrial gene cluster. To evaluate the role of type 3 fimbriae in virulence, a type 3 fimbria mutant and a type 1 and type 3 fimbria double mutant was constructed. Type 3 fimbria expression was found to strongly promote biofilm formation. However, the fimbria mutants were as effective at colonizing the intestine as the wild type, and their virulence was not attenuated in a lung infection model. Also, in a urinary tract infection model, type 3 fimbriae did not influence the virulence, whereas type 1 fimbriae were verified as an essential virulence factor. Thus, type 3 fimbriae were established not to be a virulence factor in uncomplicated K. pneumoniae infections. However, since type 3 fimbriae promote biofilm formation, their role in development of infections in catheterized patients needs to be elucidated.

Download Full-text

Role of MrkJ, a Phosphodiesterase, in Type 3 Fimbrial Expression and Biofilm Formation in Klebsiella pneumoniae

Journal of Bacteriology ◽

10.1128/jb.00304-10 ◽

2010 ◽

Vol 192 (15) ◽

pp. 3944-3950 ◽

Cited By ~ 42

Author(s):

Jeremiah G. Johnson ◽

Steven Clegg

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Surface Expression ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Bacterial Genes ◽

Type 3

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that has been shown to adhere to human extracellular matrices using the type 3 fimbriae. Introduction of plasmids carrying genes known to alter intracellular cyclic-di-GMP pools in Vibrio parahaemolyticus revealed that these genes also altered type 3 fimbrial surface expression in K. pneumoniae. Immediately adjacent to the type 3 fimbrial gene cluster is a gene, mrkJ, that is related to a family of bacterial genes encoding phosphodiesterases. We identify here a role for MrkJ, a functional phosphodiesterase exhibiting homology to EAL domain-containing proteins, in controlling type 3 fimbria production and biofilm formation in K. pneumoniae. Deletion of mrkJ resulted in an increase in type 3 fimbria production and biofilm formation as a result of the accumulation of intracellular cyclic-di-GMP. This gene was shown to encode a functional phosphodiesterase via restoration of motility in a V. parahaemolyticus strain previously shown to accumulate cyclic-di-GMP and in vitro using phosphodiesterase activity assays. The effect of the mrkJ mutation on type 3 fimbrial expression was shown to be at the level of mrkA gene transcription by using quantitative reverse transcription-PCR. These results reveal a previously unknown role for cyclic-di-GMP in type 3 fimbrial production.

Download Full-text

Biofilm Formation In Vitro and Virulence In Vivo of Mutants of Klebsiella pneumoniae

Infection and Immunity ◽

10.1128/iai.72.8.4888-4890.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4888-4890 ◽

Cited By ~ 26

Author(s):

Heather F. Lavender ◽

Jennifer R. Jagnow ◽

Steven Clegg

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

In Vitro Techniques ◽

Early Stages ◽

Airway Infections

ABSTRACT One of the early stages of Klebsiella pneumoniae airway infections may involve biofilm formation. Bacterial biofilm formation is frequently investigated using in vitro techniques that facilitate identification and analysis of individual genes. We investigated the correlation between K. pneumoniae biofilm formation in vitro and ability to cause infection in vivo following construction of a bank of mini-Tn5 mutants.

Download Full-text

The role of pgaC in Klebsiella pneumoniae virulence and biofilm formation

Microbial Pathogenesis ◽

10.1016/j.micpath.2014.11.005 ◽

2014 ◽

Vol 77 ◽

pp. 89-99 ◽

Cited By ~ 25

Author(s):

Kuang-Ming Chen ◽

Ming-Ko Chiang ◽

Meilin Wang ◽

Han-Chen Ho ◽

Min-Chi Lu ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation

Download Full-text

C3 Promotes Clearance of Klebsiella pneumoniae by A549 Epithelial Cells

Infection and Immunity ◽

10.1128/iai.72.3.1767-1774.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1767-1774 ◽

Cited By ~ 26

Author(s):

Beatriz de Astorza ◽

Guadalupe Cortés ◽

Catalina Crespí ◽

Carles Saus ◽

José María Rojo ◽

...

Keyword(s):

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Alveolar Epithelial Cells ◽

Clinical Isolates ◽

Complement Components ◽

Innate Defense ◽

Alveolar Epithelial

ABSTRACT The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

Download Full-text

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

International Journal of Molecular Sciences ◽

10.3390/ijms222112084 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12084

Author(s):

Michał Śmiga ◽

John W. Smalley ◽

Paulina Ślęzak ◽

Jason L. Brown ◽

Klaudia Siemińska ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Disease Process ◽

Human Keratinocytes ◽

Bacterial Biofilm ◽

Pathogenic Potential ◽

Glycated Proteins ◽

Sds Page

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

Download Full-text

The Antibiofilm Activity of Dual-Function Tail Tubular Protein B from KP32 Phage

10.20944/preprints201803.0075.v2 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ewa Brzozowska ◽

Anna Pyra ◽

Krzysztof Pawlik ◽

Sabina Górska ◽

Andrzej Gamian

Keyword(s):

Klebsiella Pneumoniae ◽

Enterococcus Faecalis ◽

Biofilm Formation ◽

Hydrolytic Activity ◽

Bacterial Biofilm ◽

Dual Function ◽

Antibiofilm Activity ◽

Synergistic Activity ◽

Antibiotic Action ◽

Phage Protein

Background: Dual function tail tubular proteins (TTP) belonging to the lytic bacteriophages are the interesting group of biologically active enzymes. Surprisingly, apart from their structural function, they are also polysaccharide hydrolyzes destroying bacterial extracellular components. One of the representatives of this group is TTPB from Klebsiella pneumoniae phage – KP32. TTPB hydrolyzes exopolysaccharide (EPS) of Klebsiella pneumoniae and Enterococcus faecalis strain. This depolymerizing feature was associated with the activity to prevent bacterial biofilm formation. TTPB can inhibit biofilm formation by K. pneumoniae, Enterobacter cloacae, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Moreover, synergistic activity with antibiotic action has been observed, most likely due to depolymerases’ facilitation of contact of antibiotic with bacterial cells. Methods: TTPB was overexpressed in E coli system, purified and tested towards the bacterial strains using agar overlay method. The hydrolytic activity of TTPB was performed using EPSs of K. pneumoniae PCM2713 and E. cloacae ATCC 13047 as the substrates. Next, we determined the reducing sugar (RS) levels in the TTPB/EPS mixtures, regarding the RS amount obtained after acidic hydrolysis. The antibiofilm activity of TTPB has been set down on bacterial biofilm using a biochemical method. Finally, we have demonstrated the synergistic activity of TTPB with kanamycin. Results: For the first time, the hydrolytic activity of TTPB towards bacterial EPSs has been shown. TTPB releases about a half of the whole RS amount of EPSs belonging to K. pneumoniae PCM 2713 and E. cloacae ATCC 13047 strains. 1.12 µM of the phage protein reduces biofilm of both strains by over 60%. Destroying the bacterial biofilm the phage protein improves the antibiotic action increasing kanamycin effectiveness up to four times.

Download Full-text

The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0497 ◽

2021 ◽

pp. 1-15

Author(s):

Akrm Ghergab ◽

Nisha Mohanan ◽

Grace Saliga ◽

Ann Karen C. Brassinga ◽

David Levin ◽

...

Keyword(s):

Biofilm Formation ◽

Acanthamoeba Castellanii ◽

Pseudomonas Chlororaphis ◽

Enhanced Sensitivity ◽

Hostile Environments ◽

The Impact ◽

Environmental Fitness ◽

Root Attachment

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Download Full-text