Ildikó Kustos

Bacterial outer membrane protein analysis by electrophoresis and microchip technology

Outer membrane proteins are indispensable components of bacterial cells and participate in several relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity. Furthermore, the effects of various factors on outer membrane protein expression, such as antibiotic treatment, mutation, changes in the environment, lipopolysaccharide modification and biofilm formation, have been analyzed. Traditionally, the outer membrane protein profile determination was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Converting this technique to capillary electrophoresis format resulted in faster separation, lower sample consumption and automation. Coupling capillary electrophoresis with mass spectrometry enabled the fast identification of bacterial proteins, while immediate quantitative analysis permitted the determination of up- and downregulation of certain outer membrane proteins. Adapting capillary electrophoresis to microchip format ensured a further ten- to 100-fold decrease in separation time. Application of different separation techniques combined with various sensitive detector systems has ensured further opportunities in the field of high-throughput bacterial protein analysis. This review provides an overview using selected examples of outer membrane proteins and the development and application of the electrophoretic and microchip technologies for the analysis of these proteins.

Molecular Epidemiology of VIM-4 Metallo-β-Lactamase-Producing Pseudomonas sp. Isolates in Hungary

ABSTRACT VIM metallo-β-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.

Antifungal unsaturated cyclic Mannich ketones and amino alcohols : Study of mechanism of action

The antifungal activity of some known unsaturated Mannich ketones and their amino alcohols has been reported and the mechanism of antifungal action has been studied. The inhibition of the fungal ergosterol, chitin, protein synthesis and pseudohypha formation was investigated. According to our results, Mannich ketones can influence the development of pseudohyphae of Candida albicans strains. In addition, they are able to induce significant changes in the protein composition of this fungal strain. Some of our Mannich ketones have shown inhibitory effect on the fungal chitin synthase enzyme. QSAR studies were also carried out.

Effect of Antibiotics on Cell Surface Hydrophobicity of Bacteria Causing Orthopedic Wound Infections

Background: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant. Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors. Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants. Methods: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test. Results were compared to the hydrophobicity of three Hungarian standard bacterial strains. The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin) – applied most often in our Department for prophylaxis and treatment of patients – were analyzed. Results: The cell surface hydrophobicity of certain strains showed considerable changes after antibiotic treatment. These alterations indicated the decrease in hydrophobicity. Supra-inhibitory concentrations (2× minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5× MIC) levels. Conclusions: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains. These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients.

Capillary electrophoretic analysis of wild type and mutant Proteus penneri outer membrane proteins

In this study the virulence factors, outer membrane proteins (OMP), lipopolysaccharides (LPS), hemolysin, and the in vivo and in vitro virulence of wild‐type Proteus penneri 357 and its two isogenic mutant variants — a transposon and a spontaneous mutant — were examined. The OMPs of these variants were analyzed by a new and fast technique, “dynamic sieving” capillary electrophoresis (CE). The OMP profiles were dominated by two peaks (39 and 43 kDa). In the P. penneri clone examined, both the transposon and the spontaneous mutations induced significant changes in the OMP patterns (in the relative percentage of the dominant proteins). CE was suitable for the comparative analysis of bacterial protein patterns in the genetic variants of this strain, and provided valuable results in connection with the bacteriological virulence. The LPS composition of the genetic variants also showed alterations. The wild type of P. penneri 357 showed a typical ladder pattern, an “S” form, and the mutants possessed “R” LPS patterns (only few bands) in the gels. In the bacteriological virulence tests the wild type of P. penneri 357 was virulent in the in vivo, and toxic in the in vitro assays, while both mutants showed neither toxicity nor pathogenicity.

Characterisation of protein composition and detection of IgA in cervicovaginal fluid by microchip technology.

In this paper the application of microchip electrophoresis to examine the protein profile of cervicovaginal fluid and the detection of IgA heavy and light chains is presented. This method is a fast growing field of technology and ensures high-speed analysis requiring only microliters of sample. Proteins with wide range of molecular masses could be separated within 1 min. Cervicovaginal specimens of healthy women showed a complex protein pattern-containing several peaks in the 15-70 kDa region. sIgA is considered to be an important protein constituent of all mucosal surfaces. Detection of sIgA in cervicovaginal samples was achievable by microchip technology. Under reduced circumstances (induced by mercaptoethanol, a component of the denaturating solution) the disulfide bonds connecting IgA heavy and light chains are broken up and chains can be detected as separate peaks during electrophoresis. In 82.5% of the cases only the light chain of IgA could be detected in the clinical samples. The intact IgA heavy chain could be demonstrated in only 12.5% of the cases. Based on our data some conclusions were provided about the correlation of these patterns with the age of patients, pH of the cervicovaginal fluid, operations performed before sample collection and usage of oral contraceptives.

Effect of antibiotic treatment on bacterial attachment to a DePuy Enduron orthopedic implant.

Background: The increasing incidence of bacterial infections in orthopedic surgery might be related to the increasing application of artificial devices. In most cases, bacteria multiply on the surface of implants in biofilms. Poor penetration of antibiotics, frequent necessity of prosthesis removal, chronic processes and financial costs emphasize the significance of preventive measures. Method: Adhesion of bacterial strains (two Staphylococcus aureus, two coagulase-negative staphylococci and two Pseudomonas aeruginosa strains isolated from orthopedic patients’ wounds) to the surface of a polyethylene cupwas investigated using an ultrasonic method. Results were compared to the adhesive ability of three Hungarian standard strains. The effect of antibiotic treatment (cefuroxime, cefotaxime, amoxicillin with clavulanic acid and amikacin) has been examined. Results: The staphylococcal strains showed significantly higher adhesive ability than Pseudomonas strains. Antibiotic treatment significantly reduced the attachment of bacteria. The higher the concentration of the antibiotics, the higher was the decrease in bacterial adhesion. Conclusions: Antibiotic prophylaxis was proven to be effective against bacterial adhesion, and, if applied at the proper time at the highest tolerable dose, it might prevent the formation of biofilms.

Antifungal Activity of Fused Mannich Ketones Triggers an Oxidative Stress Response and Is Cap1-Dependent in Candida albicans

We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C. albicans and the closely related C. parapsilosis and C. krusei while having reduced efficacy toward C. glabrata and almost no efficacy against Aspergillus sp. Transcript profiling of C. albicans cells exposed for 30 or 60 min to 2-(morpholinomethyl)-1-indanone, a representative FMK with a five-membered saturated ring, revealed a transcriptional response typical of oxidative stress and similar to that of a C. albicans Cap1 transcriptional activator. Consistently, C. albicans lacking the CAP1 gene was hypersensitive to this FMK, while C. albicans strains overexpressing CAP1 had decreased sensitivity to 2-(morpholinomethyl)-1-indanone. Quantitative structure–activity relationship studies revealed a correlation of antifungal potency and the energy of the lowest unoccupied molecular orbital of FMKs and unsaturated Mannich ketones thereby implicating redox cycling-mediated oxidative stress as a mechanism of action. This conclusion was further supported by the loss of antifungal activity upon conversion of representative FMKs to aminoalcohols that were unable to participate in redox cycles.

Effect of Spontaneous and Induced Mutations on Outer Membrane Proteins and Lipopolysaccharides of Proteus Penneri Strain 357

Capillary electrophoresis was suitable for the comparative analysis of the OMPs of the wild type and mutant variants of P. penneri strain 357. The patterns were reproducible and characteristic for the different genetic types examined. OMP patterns of the two mutants showed significant changes compared to the wild type. Examination of the LPS molecules showed also altered patterns in the mutants (S-R mutation). One part of alterations in OMP profiles might be explained by the specific interaction of OMP and LPS components;the proper trimerisation and function of porin proteins requires specific LPS structure8. Beside that the mutants showed neither toxicity in vitro, nor pathogenicity in vivo in the virulence assays. So the electrophoretic characterisation of both OMP and LPS composition can significantly improve the sensitivity of classical methods extensively used to identify and characterise bacterial strains.

Application of chip-based flow cytometry for amphotericin B and fluconazole susceptibility testing on Candida strains

Chip-based flow cytometry is a rather new method that offers an easy, fast opportunity for examination of yeasts, such as Candida cells. In our study cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against antifungal agents, amphotericin B and fluconazole. We found this technology to be suitable for the detection of Candida cells, for the differentiation between dead and living cells, and for the determination of amphotericin B and fluconazole susceptibility of different Candida strains (Bouquet et al., Mycoses 55:e90-e96, 2012).