Recombinant Corynebacterium urealyticum Nucleoside diphosphate kinase (ndk)

What is Corynebacterium urealyticum and why is it significant in clinical microbiology?

Corynebacterium urealyticum is a Gram-positive, slow-growing, lipophilic, multi-drug resistant bacterium with diphtheroid morphology. It possesses strong urease activity, which plays a critical role in its pathogenicity. The organism has been reported as an opportunistic nosocomial pathogen causing various diseases including cystitis, pyelonephritis, and bacteremia . Its clinical significance lies in its ability to cause complicated urinary tract infections (UTIs), particularly in immunocompromised patients and those with predisposing factors such as urological manipulations or kidney transplantation . Its multi-drug resistant nature presents significant treatment challenges, with 97.5% of isolates demonstrating resistance to multiple antibiotics .

What are the taxonomic and phylogenetic characteristics of C. urealyticum?

C. urealyticum was originally characterized based on its biochemical properties, including its inability to produce acid from carbohydrates and its ability to hydrolyze urea. Its cell wall peptidoglycan contains meso-diaminopimelic acid, with arabinose and galactose as major cell wall sugars. The organism also contains tuberculostearic acid and short-chain mycolic acids similar to Mycobacterium tuberculosis . Phylogenetically, DNA-DNA hybridization studies and 16S ribosomal RNA sequence analysis have demonstrated a close relationship between C. urealyticum and C. jeikeium . The C. urealyticum DSM7109 chromosome is smaller than other pathogenic corynebacteria, suggesting further gene reduction during its evolution .

What are the optimal conditions for isolating and identifying C. urealyticum in laboratory settings?

C. urealyticum isolates are frequently missed in routine urine cultures as they do not grow well after overnight incubation. For successful isolation, samples should be incubated for 48 hours at 35-37°C, preferably in 10% CO2, on blood agar. The resulting colonies are characteristically pinpoint, whitish, opaque, smooth, convex, circular, and non-hemolytic .

Identification can be performed through:

• Phenotypic studies using home-made media or commercial systems like API Coryne

• Molecular methods including PCR amplification

• MALDI-TOF mass spectrometry (for rapid identification)

The identification of C. urealyticum presents significant challenges for laboratories. Proper identification is crucial to differentiate contamination or colonization from actual infection . Selective media containing antibiotics, Tween-80, and urea can facilitate C. urealyticum identification and growth, as demonstrated in kidney transplant studies .

What methodological approaches can be used to express and purify recombinant proteins from C. urealyticum?

While the search results don't provide specific protocols for C. urealyticum ndk expression, researchers should consider:

• Vector selection: Based on research with related Corynebacterium species, E. coli expression systems with T7 promoter-based vectors (pET series) may be suitable.

• Expression conditions: Since C. urealyticum is slow-growing and has specific nutritional requirements, expression in heterologous hosts like E. coli BL21(DE3) with optimization of temperature (likely 25-30°C) and induction parameters is recommended.

• Protein purification: Affinity chromatography using His-tag or GST-tag fusion proteins followed by size exclusion chromatography would be appropriate for obtaining pure recombinant ndk protein.

• Activity assessment: Nucleoside diphosphate kinase activity can be measured through coupled enzyme assays monitoring the transfer of phosphate groups between nucleoside diphosphates and triphosphates.

What are the virulence factors of C. urealyticum and how do they contribute to pathogenicity?

The primary virulence factor of C. urealyticum is its urease activity, which hydrolyzes urea to ammonia, increasing urinary pH and leading to the formation of struvite crystals and biofilms. Additional virulence factors include:

• Surface proteins: The genome sequence of C. urealyticum DSM7109 contains genes for two putative surface proteins (surA and surB) that display internal repeats in their amino acid sequences and may play important roles in UTIs, particularly in catheterized patients .

• Cell surface hydrophobicity: This property promotes adherence to catheter materials such as polyvinyl chloride, Teflon, and Teflon-coated rubber, facilitating biofilm development on solid surfaces .

• Adherence capacity: In vitro studies have demonstrated tropism of C. urealyticum towards LNCaP (lymph node carcinoma of the prostate) human cell line, suggesting potential involvement in prostatitis .

How can researchers effectively model C. urealyticum infections in experimental settings?

Based on the available literature, researchers investigating C. urealyticum infections should consider:

• In vitro cell adhesion models: Studies have demonstrated successful use of LNCaP (prostate) and HeLa cell lines to study C. urealyticum adhesion and tropism . These models can be used to:

  • Quantify bacterial adhesion through MTT assays

  • Evaluate biofilm formation on various materials

  • Test the effects of potential inhibitors like acetohydroxamic acid

• Animal models: While specific animal models are not detailed in the search results, researchers might consider urinary tract infection models in immunocompromised mice, similar to those used for other urinary pathogens.

• Ex vivo models: Using urinary catheters and medical devices in controlled laboratory conditions to study biofilm formation and encrustation processes.

What is the significance of C. urealyticum's tropism towards prostate cells and what methodological approaches can investigate this phenomenon?

Preliminary in vitro studies have demonstrated tropism of C. urealyticum towards the LNCaP human prostate cell line, suggesting this organism may be a candidate for chronic idiopathic prostatitis . This finding is significant as only one clinical case of prostatitis caused by C. urealyticum has been reported, despite various coryneform bacteria being found in the male genital tract of prostatitis patients.

To investigate this phenomenon, researchers may employ:

• Cell adhesion assays: Quantitative measurement of bacterial attachment to LNCaP cells compared to control cell lines.

• Growth curve analysis: Determining bacterial growth dynamics in the presence of prostate cells.

• MTT viability assays: Assessing the cytotoxic effects of C. urealyticum on prostate cells.

• Biofilm formation assessment: Evaluating the ability of C. urealyticum to form biofilms in the presence of prostate-derived factors.

• Modulation experiments: Testing the effect of urease inhibitors like acetohydroxamic acid (AHA) on bacterial adhesion and biofilm formation .

What are the genetic mechanisms of antimicrobial resistance in C. urealyticum?

C. urealyticum exhibits extensive antimicrobial resistance with multiple genetic mechanisms identified:

• β-lactam resistance: Associated with the presence of the blaA gene encoding a class A β-lactamase, resulting in 100% resistance to ampicillin .

• Macrolide resistance: 95% of strains show resistance to erythromycin due to the ermX gene. Sequencing of this gene in resistant strains showed 95% identity with the ermX gene of reference strain C. urealyticum DSM 7109 .

• Fluoroquinolone resistance: 95% of isolates demonstrate resistance to levofloxacin, primarily due to mutations in the QRDR region of the gyrA gene. The most common mutations are:

  • Double mutation Ser-90→Val and Asp-94→Tyr (22 isolates)

  • Asp-94→Ala replacement (3 isolates)

  • Single Ser-90→Val mutation (3 isolates)

• Rifampicin resistance: Associated with non-conservative changes in Ser-444 or Gln-511 in the β subunit of RNA polymerase (RpoB) .

• Aminoglycoside resistance: Low-level gentamicin resistance has been linked to the presence of an aac(3)-XI orthologous gene encoding an aminoglycoside 3-N acetyltransferase .

• Tetracycline resistance: Attributed to an efflux-mediated mechanism .

What methodological approaches can be used to detect and characterize antimicrobial resistance in C. urealyticum?

Researchers investigating antimicrobial resistance in C. urealyticum should consider the following methods:

• Antimicrobial susceptibility testing:

  • Epsilometer test (E-test) for MIC determination

  • Disk diffusion methods with appropriate interpretation criteria

• Molecular detection of resistance genes:

  • PCR amplification of known resistance genes (ermX, blaA)

  • Sequencing of target genes (gyrA, rpoB) to identify mutations

  • Whole genome sequencing to identify novel resistance determinants

• Clonal relationship analysis:

  • Pulsed-field gel electrophoresis (PFGE) to determine strain relatedness

  • MLST (Multi-locus sequence typing) for epidemiological studies

• Phenotypic confirmation:

  • β-lactamase production assays

  • Efflux pump inhibition tests for tetracycline resistance

Research has shown that C. urealyticum isolates typically display diverse PFGE patterns, indicating they are not clonally related but rather cause sporadic infections .

What therapeutic strategies are effective against multidrug-resistant C. urealyticum infections?

Based on antimicrobial susceptibility patterns, effective therapeutic approaches include:

• Glycopeptide antibiotics: All strains remain susceptible to vancomycin and teicoplanin, making these first-line options for serious infections .

• Oxazolidinones: Linezolid shows good activity with MIC90 = 1 mg/L .

• Rifamycins: Rifampicin demonstrates good activity (MIC90 = 0.4 mg/L) against most strains, though resistance can develop .

• Combination therapy: For severe infections, particularly encrusted cystitis or pyelitis, combination antibiotic therapy may be warranted.

• Urease inhibitors: Acetohydroxamic acid (AHA) not only blocks urease activity to facilitate the removal of encrustations but also modulates pathogenic mechanisms .

• Surgical intervention: In severe cases of encrusted cystitis or pyelitis, surgical removal of encrustations may be necessary alongside antimicrobial therapy .

What is the potential of nucleoside diphosphate kinase (ndk) as a therapeutic target or research tool?

While the search results don't specifically address C. urealyticum ndk, based on knowledge of ndk in other bacterial species:

• Enzyme inhibition strategy: As ndk catalyzes the transfer of phosphate groups between nucleoside diphosphates and triphosphates, it plays a crucial role in nucleotide metabolism. Selective inhibitors could potentially disrupt bacterial replication and survival.

• Structural biology applications: Recombinant ndk could be used for crystallization studies to determine three-dimensional structure, facilitating structure-based drug design.

• Immunological studies: Purified recombinant ndk could be used to develop serological tests or as a candidate antigen for vaccine development.

• Metabolic pathway analysis: Studying ndk function could provide insights into nucleotide metabolism pathways specific to C. urealyticum, potentially revealing unique aspects of its physiology.