Urinary tract infections caused by staphylococcus aureus DNA in comparison to the candida albicans DNA.

BACKGROUND: Bacterial DNA released upon bacterial autolysis or killed by antibiotics, hence, many inflammatogenic reactions will be established leading to serious tissue damage. AIM: the present work aimed to elucidate the histopathological changes caused by prokaryotic (bacterial) DNA and eukaryotic (candidal) DNA.
MATERIALS AND METHODS: twenty one Staphylococcus aureus and 36 Candida albicans isolates were isolated from UTI patients. Viable cells and DNA of the highest antibiotic sensitive isolates were injected, intraurethraly, in mice. Results were evaluated via histopathological examination.
RESULTS: Mildest reactions were obtained from mice challenged with viable C. albicans compared with those challenged with viable S. aureus. Dose-dependent histological changes were observed for both eukaryotic and prokaryotic DNA. However, the eukaryotic C. albicans DNA developed less intense histological changes than S. aureus DNA.
CONCLUSION: microbial DNA has the ability to cause damage in murine renal system. Nevertheless, bacterial DNA caused more intense damage than candidal DNA.

Introduction

Microbial components signal the presence of foreign pathogens in the innate immune system. They are key players in infectious diseases and implicate toll-like receptors (TLR) in the activation of inflammation[1].

Bacterial DNA causes potent immune response stimulation. The first report on the immunostimulatory properties of bacterial DNA date back to Tokunaga and colleagues in 1984 due to the presence of unmethylated CpG dinucleotides in a particular base sequence context termed “CpG motif”. Importantly, the immunostimulatory activity is found in bacterial, yeast, viruses, insect and nematode DNA[2]. In contrast, eukaryotic DNA is nonstimulatory; this lack of immunostimulatory potential has been explained by CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake in combined[3].

Microbial DNA activates innate immune cells via TLR 9 leading to secretion of pro-inflammatory cytokines and directs the development of adaptive immunity, suggesting that microbial DNA is an important virulence determinant and inflammatory stimulus during infections[4]. Deng et al.[5] reported that when the microbe degraded or autolysed for any reason such as killing by antibiotics or processing by the antigen presenting cells, the DNA will be released, hence, it will evoke the inflammatogenic reactions causing serious problems.

For the critical role of DNA in pathogenesis and the absence of in vivo study tracing the comparison between prokaryotic unmethylated DNA and eukaryotic slightly methylated DNA in the respect of the urinary system, this work aimed to conduct a comparative histopathological study between DNA extracted from S. aureus and C. albicans in urinary tract of mice.

Materials and Methods

Isolation and Identification

Mid-stream urine specimens were collected from 308 patients (15-50 years), presented with urinary tract infections visiting Baghdad hospitals.

All specimens were inoculated on mannitol salt agar and sabouraud dextrose agar, incubated at 37°C and 30°C, respectively for 24hrs. Staphylococci were identified depending on biochemical tests according to Forbes et al.[6]. The classification was achieved according to Holt et al.[7]. API-Staph system was employed to confirm the identification. Candida albicans was identified depending on the morphological features on culture medium and germ tube formation in addition to API-Candida system.

Antibiotic Susceptibility

The modified Kirby-Bauer method[8] was used to investigate the antibiotic susceptibility against amoxicillin, ampicillin, cefotaxime, methicillin, oxacillin, and vancomycin. A standard S. aureus strain (S. aureus ATCC 6538P) was used as the quality control strain.

DNA Extraction

Staphylococcus aureus DNA were prepared using a Wizard genomic DNA purification kit (Promega). The method of Harju et al.[9] was followed to extract the DNA from Candida albicans. DNA preparations were stored at 2-8°C.

Animals

Female white BALB/C mice (age 6-8 weeks, weight 20-25 gm) were used in this study. Mice were housed in plastic cages and fed ad libitum with a conventional diet.

Injection Protocol

The bladder was emptied from urine by pressing on abdominal area. Urethra and surrounding area were sterilized with 70% ethanol then a polyethylene tube (0.6 mm in diameter) was introduced to urinary bladder via urethra; the inoculums (1.5 × 108 CFU/ml and 1 × 108 CFU/ml for S. aureus and C. albicans, respectively) was injected by aid of this catheter. Thereafter the catheter was withdrawn immediately, animals were returned to their cages with their lower end directed upward to avoid effusion of the inoculum outside[10].

Mice were divided into fourteen groups, two animals per group:

First, second, third, fourth and fifth groups were injected with 0.1ml of S. aureus S11 DNA at concentrations 10, 20, 30, 40 and 50 μg/100μl, respectively.

Sixth, seventh, eighth, ninth and tenth groups were injected with 0.1ml of C. albicans C3 DNA at different concentrations 10, 20, 30, 40 and 50 μg/100μl respectively.

Eleventh and Twelfth groups were injected with 0.1ml of S. aureus S11 and C. albicans C3 suspensions, respectively.

Thirteenth and fourteenth groups (control groups) were injected with normal saline and TE buffer respectively.

All groups were injected with two repeat to each injection and kept in their cages without water for 24hrs. 3 days later, they were sacrificed; kidneys and bladders were removed for histopathological study.

Results

Out of 72 staphylococcal isolates, 51 were identified as coagulase negative staphylococci and 21 as S. aureus. 36 specimens were identified as C. albicans. The isolates S. aureus S11 and C. albicans C3 showed the highest antibiotic susceptiblity, hence, they were selected for in vivo study.

Histopathology

No histological changes were observed in kidney and bladder of control mice as shown in Figure 1.

Fig. 1

a: histological section of control mouse kidney 3 days after challenged with 0.1ml of saline shows a normal structure appearance of glomeruli (G) and renal tubules (T). b: The histological section of control mouse urinary bladder 3 days after challenged with 0.1ml of saline shows a normal structure appearance of urinary epithelial lining mucosa (E) and subepithelial lining layer (SE). H & E (X200).

Histopathological examination of kidney from mice challenged with viable S. aureus S11 revealed congestion, inflammatory cells infiltration and degenerative changes of renal tubules (Figure 2a), while its bladder suffered from edema, moderate inflammatory cells infiltration and focal mucosal surface epithelial ulceration (Figure 2b). Whereas those of mice challenged with viable C. albicans C11 showed congestion (Figure 2c). The bladder developed mild infiltration of inflammatory cells with edema in subepithelial lining layer (Figure 2d).

Fig. 2

a: Histological section of mouse kidney 3 days after challenged with 0.1 ml of 1.5 × 108 cfu/ml of S. aureus S11 shows congestion (CO) ), inflammatory cells infiltration (IC) and degenerative changes of renal tubules (D). b: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 1.5 ×108 cfu/ml of S. aureus S11 shows edema (O) with moderate inflammatory cells infiltration (IC) and focal mucosal surface epithelial ulceration (FU). c: The histological section of mouse kidney 3 days after challenged with 0.1ml of 1 × 108 cfu/ml of C. albicans C3 shows congestion (CO) and no obvious histological changes. d: The histological section of mouse urinary bladder 3 days after intraurethrally challenged with 0.1ml of 1×108 cfu/ml of C. albicans C3 shows mild infiltration of inflammatory cells (IC) with edema (O) in subepithelial lining layer. H & E (X200).

Kidney challenged with 50μg/100μl of S. aureus S11 DNA showed congestion, degenerative and necrosis of renal tubule with severe infiltration of inflammatory cells, the bladder revealed damage of mucosal wall with severe inflammatory cells infiltration (Figure 3a, b). At 40 μg/100μl histological sections of kidney showed congestion of blood vessel with degenerative changes of renal tubules, the bladder developed mild edema and infiltration of inflammatory cells with sloughing of mucosal epithelial surface (Figure 3c, d). While at 30 μg/100μl histological sections of the kidney revealed congestion and mild degenerative changes of renal tubules with mild inflammatory cells infiltration, the bladder showed edema with infiltration of inflammatory cells (Figure 3e, f). At 20 μg/100μl and 10 μg/100μl, histological sections showed normal structure appearance of the kidney and the bladder.

Fig. 3

a: The histological section of mouse kidney 3 days after challenged with 0.1ml of 50 μg/100 μl of S. aureus S11 DNA showing congestion (CO), degenerative and necrosis (DN) of renal tubule with severe infiltration of inflammatory cells (IC). b: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 50 μg/100 μl of S. aureus S11 DNA showing damage of mucosal wall (D) with severe inflammatory cells infiltration (IC). c: The histological section of mouse kidney 3 days after challenged with 0.1ml of 40 μg/100 μl of S. aureus S11 DNA showing congestion (CO) of blood vessel with degenerative changes (D) of renal tubules. d: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 40 μg/100 μl of S. aureus S11 DNA showing mild edema (O) and infiltration of inflammatory cells (IC) with sloughing (S) of mucosal epithelial surface. e: The histological section of mouse kidney 3 days after challenged with 0.1ml of 30 μg/100 μl of S. aureus S11 DNA showing congestion (CO) and mild degenerative changes (D) of renal tubules with mild inflammatory cells infiltration (IC). f: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 30 μg/100 μl of S. aureus S11 DNA showing edema (O) with infiltration of inflammatory cells (IC). g: The histological section of mouse kidney 3 days after challenged with 0.1ml of 50 μg/100 μl of C. albicans C3 DNA showing congestion (CO) with moderate inflammatory cells infiltration (IC). h: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 50 μg/100 μl of C. albicans C3 DNA showing edema (O) with mild inflammatory cells infiltration (IC). i: The histological section of mouse kidney 3 days after challenged with 0.1ml of 40 μg/100 μl of C. albicans C3 DNA showing congestion (CO) of blood vessels. j: The histological section of mouse urinary bladder 3 days after challenged with 0.1ml of 40 μg/100 μl of C. albicans C3 DNA showing mild inflammatory cells (IC) infiltration and edema (O). H & E (X200).

Sections of kidney challenged with 50 μg/100μl of C. albicans C3 DNA showed congestion with moderate inflammatory cells infiltration, the bladder revealed edema with mild inflammatory cells infiltration (Figure 3g, h). At 40 μg/100μl kidney showed congestion of blood vessels, the bladder developed mild inflammatory cells infiltration and edema (Figure 3i, j). While at 10, 20 and 30 μg/100μl, histological sections showed normal kidney and bladder structure.

Discussion

Tissue damage was developed with features relatively less intense in mice challenged with C. albicans than those whom challenged with S. aureus. That may be due various virulence factors produced by S. aureus of[11] and C. albicans[12]. From the previous findings it was concluded that the DNA extracted from S. aureus S11 bacteria and C. albicans C3 yeast may function as a virulence factor of pathogenic microbes via histopathological effects that caused to kidneys and bladders treated by both DNA.

The inflammatory effects of DNA may result from the production of cytokines and chemokines[13]. Recognition of microbial DNA by TLR 9 in mammalian phagocytic cell triggers an immunostimulatory cascade that culminates in the maturation, differentiation, and/or proliferation of multiple cell types. Together, these cells secrete cytokines and chemokines that create proinflammatory immune stimuli and other mediators of inflammation in addition to reactive oxygen species generation[14-17]. Thus, this cell population and its soluble products are crucial to the observed inflammatory response to DNA[18], and might participate, directly or indirectly, in the pathogenesis of urinary tract infection.

Comparison between S. aureus DNA and C. albicans DNA The present study indicates that histological changes caused by eukaryotic fungal pathogen C. albicans C3 DNA were generally with potency lower than that of prokaryotic bacterial pathogen S. aureus S11 DNA.

They were of approximately similar GC content DNA organism (32.5 % GC of S. aureus DNA and 35.1% GC of C. albicans DNA) in order to show that the differences observed in histological changes between both were not due to the difference in frequency of CG (fCG). As that the dinucleotide (CG) showed a frequency as expected from the individual G+C content and that an increase in fCG went along with increased immunostimulation[19].

Candida albicans DNA have mitogenic properties for immune cells that are similar to those of bacterial DNA, which correlated with the presence of nonmethylated CG dinucleotides and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. albicans. In addition C. albicans DNA may contain as yet unidentified immunostimulatory structure other than CpG motifs that may be present at a higher frequency in this organism[20].

There are several explanations why C. albicans DNA may not be a strong inflammatory agent: 1) C. albicans DNA contains methylated DNA which diminishes the activity of CpG DNA via interaction with TLR9[2122] ; 2) the CpG frequency of C. albicans DNA may be less than that of S. aureus and may explain the difference in the activating capabilities of these two DNA; 3) it may be that candidal DNA has inhibitory motifs as that of vertebrate DNA which inhibit the immunostimulatory function of unmethylated CpG motifs[3]. Miyazato et al.[20] hypothesized that some inhibitory effect of C. albicans DNA could interfere with the DNA stimulation. This could be due to the inhibition of DNA uptake by some molecules or inhibitory DNA sequences that can bind TLR9 sufficiently and neutralized the CpG induced activation of immune cells. Finally, alternatively, stimulation by unmethylated CpG motifs require optimal flanking sequences and/or depend on localization of the unmethylated CpG motifs within the genomic DNA sequence. Hence, the high potency of bacterial DNA probably reflects the presence of optimal flanking sequences around CpG motifs which is known that the optimal flanking sequences higher for bacterial than for eukaryotic DNA[14]. In this respect, the optimal flanking sequences may be higher for S. aureus than for C. albicans DNA.

Effect of DNA concentration on histological changes

DNA from S. aureus S11 induced significant histopathological changes starting from 10 μg/100 μl and increasing up to 50 μg/100 μl. DNA from C. albicans C3 was effective at concentrations above 30 μg/100 μl but developed less changes than observed with S. aureus S11 DNA. Most of these results agreed with many studies correlated with the important role of DNA in the pathogenesis of microbes[514162021].

The histological changes severity evaluated with regard to the extent of the inflammation and judged on an arbitrary scale (Figure 4); from Grade 0 (no signs of inflammation), Grade 1 (mild diffuse or focal inflammation in a single area), to grade 2 (moderate diffuse or focal inflammation), and Grade 3 (heavy diffuse and focal inflammation)[18]. Mölne et al.[18] reported that when S. aureus DNA was injected intradermally into mice, moderate to heavy inflammatory changes in skin and focal inflammation in an extended pattern were triggered. Tasaka et al.[23] mentioned that intratracheal instillation of CpG-ODN revealed acute lung injury in a dose-dependent effect.

Fig. 4

Effect of DNA concentrations on histological changes severity

In issue of periodontal disease Takeshita et al.[16] suggested that Porphyromonas gingivalis DNA may function as a virulence factor in periodontal disease through expression of inflammatory cytokine. The bacterial DNA markedly stimulated in a dose-dependent manner IL-6 production by human gingival fibroblasts. In a study done by Al-Mathkhury and Al-Zubeidy[15] , E. coli DNA caused shrinkage of glomerulus and increased capsular space, edema and inflammatory cells infiltration in kidney tissue while the urinary bladder suffered from infiltration of inflammatory cells. Anders et al.[17] found that E. coli DNA increased serum DNA autoantibodies in association with progression of glomerulonephritis.

Conclusion

Bacterial DNA has the ability to cause damage in renal tissue in a dose dependent manner. Additionally, this damage intense was more than candidal DNA. Furthermore, even though the microbe is killed or auloysed the risk of inflammation is still need to be investigated.