Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

ABSTRACT Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.

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Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces

Microbiology ◽

10.1099/mic.0.26434-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2397-2405 ◽

Cited By ~ 91

Author(s):

Jennifer Jagnow ◽

Steven Clegg

Keyword(s):

Extracellular Matrix ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Fimbrial Subunit ◽

Direct Binding ◽

Respiratory Cells ◽

Coated Surfaces ◽

Plastic Surfaces ◽

Type 3

The type 3 fimbriae of Klebsiella pneumoniae are comprised of the major fimbrial subunit (MrkA) and the adhesin (MrkD) that has previously been shown to mediate binding to collagen. The ability of adhesive and non-adhesive derivatives of K. pneumoniae to form biofilms on collagen-coated surfaces in continuous-flow chambers was investigated. Unlike biofilm formation on abiotic plastic surfaces, the presence of the MrkD adhesin was necessary for growth on collagen-coated surfaces. Fimbriate strains lacking the MrkD adhesin did not efficiently adhere to and grow on these surfaces. Similarly, purified human extracellular matrix and the extracellular matrix formed by human bronchial epithelial cells grown in vitro provided a suitable substrate for MrkD-mediated biofilm formation, whereas direct binding to the respiratory cells was not observed. Type 3 fimbriae may therefore have two roles in the early stages of adherence and growth on in-dwelling devices such as endotracheal tubes. The MrkA polypeptide could facilitate adsorption to abiotic polymers of recently implanted devices and the MrkD adhesin could enable bacteria to adhere to and grow on polymers coated with host-derived proteins.

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An optimised GAS-pharyngeal cell biofilm model

Scientific Reports ◽

10.1038/s41598-021-87377-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Heema K. N. Vyas ◽

Jason D. McArthur ◽

Martina L. Sanderson-Smith

Keyword(s):

Crystal Violet ◽

Biofilm Formation ◽

Cell Model ◽

Matrix Material ◽

Three Dimensional ◽

Growth Period ◽

Optimal Growth ◽

Abiotic Surfaces

AbstractGroup A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

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Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

Infection and Immunity ◽

10.1128/iai.73.6.3210-3218.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3210-3218 ◽

Cited By ~ 100

Author(s):

Joseph Jurcisek ◽

Laura Greiner ◽

Hiroshi Watanabe ◽

Anthony Zaleski ◽

Michael A. Apicella ◽

...

Keyword(s):

Sialic Acid ◽

Haemophilus Influenzae ◽

Middle Ear ◽

Biofilm Formation ◽

Mammalian Host ◽

Nontypeable Haemophilus Influenzae ◽

Biofilm Production ◽

Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

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Chlorpromazine-impregnated catheters as a potential strategy to control biofilm-associated urinary tract infections

Future Microbiology ◽

10.2217/fmb-2019-0092 ◽

2019 ◽

Vol 14 (12) ◽

pp. 1023-1034 ◽

Cited By ~ 3

Author(s):

José JC Sidrim ◽

Bruno R Amando ◽

Francisco IF Gomes ◽

Marilia SMG do Amaral ◽

Paulo CP de Sousa ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Proteus Mirabilis ◽

Biofilm Formation ◽

Antibiofilm Activity ◽

Potential Strategy ◽

Tract Infections

Aim: This study proposes the impregnation of Foley catheters with chlorpromazine (CPZ) to control biofilm formation by Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae. Materials & methods: The minimum inhibitory concentrations (MICs) for CPZ and the effect of CPZ on biofilm formation were assessed. Afterward, biofilm formation and the effect of ciprofloxacin and meropenem (at MIC) on mature biofilms grown on CPZ-impregnated catheters were evaluated. Results: CPZ MIC range was 39.06–625 mg/l. CPZ significantly reduced (p < 0.05) biofilm formation in vitro and on impregnated catheters. In addition, CPZ-impregnation potentiated the antibiofilm activity of ciprofloxacin and meropenem. Conclusion: These findings bring perspectives for the use of CPZ as an adjuvant for preventing and treating catheter-associated urinary tract infections.

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Salmonella Extracellular Matrix Components Influence Biofilm Formation and Gallbladder Colonization

Infection and Immunity ◽

10.1128/iai.00532-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3243-3251 ◽

Cited By ~ 13

Author(s):

Haley E. Adcox ◽

Erin M. Vasicek ◽

Varun Dwivedi ◽

Ky V. Hoang ◽

Joanne Turner ◽

...

Keyword(s):

Extracellular Matrix ◽

Typhoid Fever ◽

Salmonella Enterica ◽

Biofilm Formation ◽

Bacterial Survival ◽

Content Type ◽

Extracellular Matrix Components ◽

Matrix Components

Salmonella enterica serovar Typhi, the causative agent of typhoid fever in humans, forms biofilms encapsulated by an extracellular matrix (ECM). Biofilms facilitate colonization and persistent infection in gallbladders of humans and mouse models of chronic carriage. Individual roles of matrix components have not been completely elucidated in vitro or in vivo . To examine individual functions, strains of Salmonella enterica serovar Typhimurium, the murine model of S . Typhi, in which various ECM genes were deleted or added, were created to examine biofilm formation, colonization, and persistence in the gallbladder. Studies show that curli contributes most significantly to biofilm formation. Expression of Vi antigen decreased biofilm formation in vitro and virulence and bacterial survival in vivo without altering the examined gallbladder pro- or anti-inflammatory cytokines. Oppositely, loss of all ECM components (Δ wcaM Δ csgA Δ yihO Δ bcsE ) increased virulence and bacterial survival in vivo and reduced gallbladder interleukin-10 (IL-10) levels. Colanic acid and curli mutants had the largest defects in biofilm-forming ability and contributed most significantly to the virulence increase of the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant strain. While the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant was not altered in resistance to complement or growth in macrophages, it attached and invaded macrophages better than the wild-type (WT) strain. These data suggest that ECM components have various levels of importance in biofilm formation and gallbladder colonization and that the ECM diminishes disseminated disease in our model, perhaps by reducing cell attachment/invasion and dampening inflammation by maintaining/inducing IL-10 production. Understanding how ECM components aid acute disease and persistence could lead to improvements in therapeutic treatment of typhoid fever patients.

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Antibacterial Activity of LCB10-0200 against Klebsiella pneumoniae

Antibiotics ◽

10.3390/antibiotics10101185 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1185

Author(s):

Sang-Hun Oh ◽

Young-Rok Kim ◽

Hee-Soo Park ◽

Kyu-Man Oh ◽

Young-Lag Cho ◽

...

Keyword(s):

Antibacterial Activity ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Antibacterial Agent ◽

In Vitro Susceptibility ◽

In Vivo Models ◽

Tract Infections

Klebsiella pneumoniae is one of the important clinical organisms that causes various infectious diseases, including urinary tract infections, necrotizing pneumonia, and surgical wound infections. The increase in the incidence of multidrug-resistance K. pneumoniae is a major problem in public healthcare. Therefore, a novel antibacterial agent is needed to treat this pathogen. Here, we studied the in vitro and in vivo activities of a novel antibiotic LCB10-0200, a siderophore-conjugated cephalosporin, against clinical isolates of K. pneumoniae. In vitro susceptibility study found that LCB10-0200 showed potent antibacterial activity against K. pneumoniae, including the beta-lactamase producing strains. The in vivo efficacy of LCB10-0200 was examined in three different mouse infection models, including systemic, thigh, and urinary tract infections. LCB10-0200 showed more potent in vivo activity than ceftazidime in the three in vivo models against the drug-susceptible and drug-resistant K. pneumoniae strains. Taken together, these results show that LCB10-0200 is a potential antibacterial agent to treat infection caused by K. pneumoniae.

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Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae

Infection and Immunity ◽

10.1128/iai.72.1.106-113.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 106-113 ◽

Cited By ~ 97

Author(s):

W. Edward Swords ◽

Miranda L. Moore ◽

Luciana Godzicki ◽

Gail Bukofzer ◽

Michael J. Mitten ◽

...

Keyword(s):

Sialic Acid ◽

Haemophilus Influenzae ◽

Middle Ear ◽

Biofilm Formation ◽

Model System ◽

Model Systems ◽

Bacterial Persistence ◽

Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

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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.

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Role of Klebsiella pneumoniae Type 1 and Type 3 Fimbriae in Colonizing Silicone Tubes Implanted into the Bladders of Mice as a Model of Catheter-Associated Urinary Tract Infections

Infection and Immunity ◽

10.1128/iai.00348-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 3009-3017 ◽

Cited By ~ 57

Author(s):

Caitlin N. Murphy ◽

Martin S. Mortensen ◽

Karen A. Krogfelt ◽

Steven Clegg

Keyword(s):

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Wild Type ◽

Content Type ◽

Tract Infections ◽

Type 3

ABSTRACTCatheter-associated urinary tract infections are biofilm-mediated infections that cause a significant economic and health burden in nosocomial environments. Using a newly developed murine model of this type of infection, we investigated the role of fimbriae in implant-associated urinary tract infections by the Gram-negative bacteriumKlebsiella pneumoniae, which is a proficient biofilm former and a commonly isolated nosocomial pathogen. Studies have shown that type 1 and type 3 fimbriae are involved in attachment and biofilm formationin vitro, and these fimbrial types are suspected to be important virulence factors during infection. To test this hypothesis, the virulence of fimbrial mutants was assessed in independent challenges in which mouse bladders were inoculated with the wild type or a fimbrial mutant and in coinfection studies in which the wild type and fimbrial mutants were inoculated together to assess the results of a direct competition in the urinary tract. Using these experiments, we were able to show that both fimbrial types serve to enhance colonization and persistence. Additionally, a double mutant had an additive colonization defect under some conditions, indicating that both fimbrial types have unique roles in the attachment and persistence in the bladder and on the implant itself. All of these mutants were outcompeted by the wild type in coinfection experiments. Using these methods, we are able to show that type 1 and type 3 fimbriae are important colonization factors in the murine urinary tract when an implanted silicone tube is present.

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Type 3 Fimbrial Shaft (MrkA) of Klebsiella pneumoniae, but Not the Fimbrial Adhesin (MrkD), Facilitates Biofilm Formation

Infection and Immunity ◽

10.1128/iai.69.9.5805-5812.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5805-5812 ◽

Cited By ~ 67

Author(s):

Jennifer Langstraat ◽

Megan Bohse ◽

Steven Clegg

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Opportunistic Infections ◽

Matrix Proteins ◽

Target Tissue ◽

Bacterial Surface ◽

Green Fluorescent ◽

Type 3

ABSTRACT Isolates of Klebsiella pneumoniae are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains ofK. pneumoniae was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of K. pneumoniaeexpressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intactmrk gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.

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