Great Plasticity in a Great Pathogen: Capsular Types, Virulence Factors and Biofilm Formation in ESBL-Producing Klebsiella pneumoniae from Pediatric Infections in Uruguay.

Klebsiella pneumoniae is widely recognized as an opportunistic hospital and community pathogen. It is one of the priority microorganisms included in the ESKAPE group, and its antibiotic resistance related to extended-spectrum ß-lactamases (ESBL) is a global public health concern. The multi-drug resistance (MDR) phenotype, in combination with pathogenicity factors, could enhance the ability of this pathogen to cause clinical infections. The aim of this study was to characterize pathogenicity factors and biofilm formation in ESBL-producing K. pneumoniae from pediatric clinical infections. Capsular types, virulence factors, and sequence types were characterized by PCR. Biofilm formation was determined by a semiquantitative microtiter technique. MDR phenotype and statistical analysis were performed. The K24 capsular type (27%), virulence factors related to iron uptake fyuA (35%) and kfuBC (27%), and sequence types ST14 (18%) and ST45 (18%) were the most frequently detected. Most of the strains were biofilm producers: weak (22%), moderate (22%), or strong (12%). In 62% of the strains, an MDR phenotype was detected. Strains with K24 capsular type showed an association with ST45 and the presence of fyuA; strains with kfuBC showed an association with moderate or strong biofilm production and belonging to ST14. Weak or no biofilm producers were associated with the absence of kfuBC. The MDR phenotype was associated with the main ESBL gene, blaCTX-M-15. The high plasticity of K. pneumoniae to acquire an MDR phenotype, in combination with the factors exposed in this report, could make it even more difficult to achieve a good clinical outcome with the available therapeutics.

Role of Proteus mirabilis flagella in biofilm formation / Papel de los flagelos de Proteus mirabilis en la formación de biofilms

Abstract Proteus mirabilis (P. mirabilis) is a common etiological agent of urinary tract infec-tions, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm genera-tion. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Resumen Proteus mirabilis (P mirabilis) es un agente etiológico común de infecciones del tracto urinario, en particular de aquellas asociadas con cateterización. P. mirabilis forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado swarming, mediado por flagelos. Hasta el momento, el papel de los flagelos en la formación de biofilms de P. mirabilis ha estado en discusión. En este estudio, se evaluó el papel de los flagelos de P. mirabilis en la formación de biofilms, utilizando una mutante isogénica generada por reemplazo alélico, incapaz de expresar flagelina. Se utilizaron diferentes enfoques, como la evaluación de la hidrofobicidad de la superficie celular, de la movilidad y la migración bacteriana sobre secciones de catéteres y medidas de biomasa y de la dinámica del biofilm mediante inmunofluorescencia y microscopia confocal, tanto en modelos estáticos como de flujo. Nuestros hallazgos indican que los flagelos de P. mirabilis desempeñan un papel en la formación de biofilms, aunque su falta no suprime por completo su generación. Asimismo, evidencian que la interferencia de la función flagelar puede contribuir a evitar la formación de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares.

Role of Proteus mirabilis flagella in biofilm formation.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Characterization of the Different Stages of Biofilm Formation and Antibiotic Susceptibility in a Clinical Acinetobacter baumannii Strain.

Acinetobacter baumannii is a relevant opportunistic pathogen, and one of the main microorganisms responsible for outbreaks in nosocomial infections worldwide. Its pathogenicity is mainly due to its resistance to multiple antibiotics and to its ability to form biofilms on abiotic surfaces. The objective of this study was to characterize the biofilm formation cycle of A. baumannii isolated from a patient in a hospital and compare its antibiotic resistance with the planktonic cells. To study biofilm formation, the classical microtiter assay was used, with crystal violet staining and optical density reading to classify the type of biofilm. Also, the effect of gentamicin and colistin on bacterial biofilm was studied with an extra step of antibiotic addition. For the characterization of the different biofilm formation stages, the strain was grown on a coverslip, and the stain was made with a mixture of fluorophores markers to visualize the biofilm with a confocal laser microscope. It was possible to differentiate the A. baumannii biofilm formation stages. Through these observations, it was possible to estimate the time elapsed between each stage. As the strain was susceptible to colistin and gentamicin, both antibiotics were evaluated after the biofilm was formed. Neither antibiotics showed an effect on the eradication of A. baumannii biofilm.

Purification of Native Flagellin.

Flagella are effective organelles of locomotion and one of several virulence factors in Proteus mirabilis. To study their properties and role in virulence, we describe a protocol to extract and purify the native flagellin of P. mirabilis. Purified flagellin can be visualized by SDS-PAGE or immunoblot and is suitable for downstream applications such as immunization.

Adherence of Proteus mirabilis to Uroepithelial Cells.

Bacterial adherence to eukaryotic cells is mediated by different adhesins that can act at different stages in bacteria-host interaction. Abundant evidence has suggested that adherence is critical for infection by bacterial pathogens. Proteus mirabilis is an opportunistic pathogen which frequently infects the human urinary tract, particularly in patients with indwelling urinary catheters. Sequencing of the genome of this pathogen has revealed the existence of a remarkable amount of complete fimbrial operons. In this chapter, we describe in vitro adherence assays of P. mirabilis to uroepithelial cells, which can provide relevant results to assess virulence of uropathogenic strains.

Magnesium-doped zinc oxide nanoparticles alter biofilm formation of Proteus mirabilis.

Aim:Proteus mirabilis biofilms colonize medical devices, and their role in microbial pathogenesis is well established. Magnesium-doped zinc oxide nanoparticles (ZnO:MgO NPs) have potential antimicrobial properties; thus, we aimed at evaluating the antibiofilm activity of ZnO:MgO NPs against P. mirabilis biofilm. Materials & methods: After synthesis and characterization of ZnO:MgO NPs and their addition to a polymer film, we evaluated the stages of P. mirabilis biofilm development over glass coverslip covered by different concentrations of ZnO:MgO NPs. Results: Low concentrations of ZnO:MgO NPs affect the development of P. mirabilis biofilm. Descriptors showed reduced values in bacterial number, bacterial volume and extracellular material. Conclusion: Our results highlight this new application of ZnO:MgO NPs as a potential antibiofilm strategy in medical devices.

Effect of different antibiotics on biofilm produced by uropathogenic Escherichia coli isolated from children with urinary tract infection.

Recurrent urinary tract infections (UTIs) occur frequently in children and women. Intracellular bacterial communities (IBCs) and biofilm formation by Escherichia coli are risk factors for recurrence. The aim of this study was to evaluate the effect of different antibiotics on biofilms by E. coli strains isolated from children with UTI and to correlate virulence factors and IBCs with biofilm formation. A total of 116 E. coli strains were tested for biofilm formation using the crystal violet microplate technique. 58.6% of the strains did not produce biofilm, while 16.4%, 18.1% and 6.8% formed weak, moderate and strong biofilms, respectively. No correlation was found between the ability to form biofilms and the presence of IBCs. Biofilm formation was significantly associated with pili P codifying genes, whereas other virulence factors were not statistically associated. Antibiotics, including ampicillin, cephalothin, ceftriaxone, ceftazidime, amikacin and ciprofloxacin, were evaluated at different concentrations after 48 h of biofilm formation. Except ampicillin, the other antibiotics tested induced a significant reduction of biofilm biomass. In the case of recurrent UTIs potentially associated with the presence of biofilm, the use of third-generation cephalosporin, fluoroquinolones and aminoglycosides could be recommended. These antibiotics demonstrated to reduce biofilm biomass produced even by resistant strains.

Fimbriae have distinguishable roles in Proteus mirabilis biofilm formation.

Proteus mirabilis is one of the most common etiological agents of complicated urinary tract infections, especially those associated with catheterization. This is related to the ability of P. mirabilis to form biofilms on different surfaces. This pathogen encodes 17 putative fimbrial operons, the highest number found in any sequenced bacterial species so far. The present study analyzed the role of four P. mirabilis fimbriae (MR/P, UCA, ATF and PMF) in biofilm formation using isogenic mutants. Experimental approaches included migration over catheter, swimming and swarming motility, the semiquantitative assay based on adhesion and crystal violet staining, and biofilm development by immunofluorescence and confocal microscopy. Different assays were performed using LB or artificial urine. Results indicated that the different fimbriae contribute to the formation of a stable and functional biofilm. Fimbriae revealed particular associated roles. First, all the mutants showed a significantly reduced ability to migrate across urinary catheter sections but neither swimming nor swarming motility were affected. However, some mutants formed smaller biofilms compared with the wild type (MRP and ATF) while others formed significantly larger biofilms (UCA and PMF) showing different bioarchitecture features. It can be concluded that P. mirabilis fimbriae have distinguishable roles in the generation of biofilms, particularly in association with catheters.