ARTICLE
Impact of in vitro clindamycin on the combination of clindamycin and ketoconazole on exopolymer of Candida spp biofilms of urogenital origin

INTRODUCTION

The vaginal mucosa has been widely used for administering antimicrobial agents to treat endogenous infections of the lower genital tract in pregnant and non-pregnant women. Candida spp. elaborates biofilms (BF), and its formation is a complex process requiring that fungal cells establish multiple interactions with the medium. They participate both in the colonization and in the progression of the disease^(1,2,3). Biofilms are surrounded by an exopolymer matrix (EP) that can restrict the activity of antibodies, the diffusion of substances and be associated with antimicrobials (AM), limiting its actions, and therefore they are significantly less susceptible to AM agents^(4,5,6). The access difficulty of general AM and particular anti-mycotic agents (AMC) to cells within the EP is phenotypically different of their corresponding planktonic or suspended cells^(7,8). Many formulas used for the empirical treatmen thave improper combinations with limited or null activity on the BF. The presence of molecules that cause its inhibition, thus eliminating the EP inducers, or by other mechanism, will allow the specific AM activity.

OBJECTIVE

To show that the activity of clindamycin used in combination with ketoconazole (CK) works on Candida albicans (CA) BF and on species of non-albicans Candida (CNA).

METHODS

We have analyzed the activity of CK regarding the adherence and dispersion of BF from eight vaginal isolates of CA and seven of CNA. The isolates were inoculated in three tubes with Sabouraud agar and a glass device to form the BF according to a known technique⁽⁹⁾.

Adherence study: Each isolate was incubated in triplicate for a six-hour period and a combination of CK from the material of ovules was added and conveniently diluted to one of the tubes of each isolate (62.5/260.4 ug/mL), considering 0 hour.

Dispersion study: The same dilution was added to the second tube after 16 hours. The third tube was used as control without AM.

CLI and KET activity: we studied the activity of CLI (62.5 ug/mL) and KET (260.4 ug/mL) separately by using a technique similar to that previously described. The AM was added after the glass device had been incubated at the 6th (adherence) and 16th hours(dispersion).

Study of the CK and BF combination activity of clinical samples: we chose the samples from seven patients showing CVV that were studied by means of the conventional techniques (pH, amine test, microscopy and cultures). We made the BF by using the technique of cell layers⁽¹⁰⁾. The CK combination was used for the study of adherence and dispersion by applying the same concentrations used for the study regarding BF of CA and CAN. All glass devices with the BF of CA, CAN submitted to the activity of CK, CLI and KET were stained with crystal violet. The devices with cell layers were stained with Gram staining. The reading was carried out with a microscope (1000x) at 24 hours, and they were photographed with a digital camera and analyzed by means of a program for studying the EPM (Optical Imaging Software).

RESULTS

Biofilms of CA and CNA with CLI and KET

The analysis of BF, submitted to CLI activity, only enabled to observe that the EPM had been dispersed, and the BF of CA and CAN were the only ones that remained. In BF with KET addition, the persistence of the EPM was seen, even though there were some cases of certain alteration of the blastospores that indicate imperfect penetration (Figure 1).

Figure 1

Biofilms of Candida albicans and non albicans Candida with the control without antimicrobial exposure, exposure to Clindamycin plus ketoconazole (CK), clindamycin alone and ketoconazole alone.

Biofilms of CVV with CK

In BF of clinical samples, neither hyphae nor mycotic elements were seen in 5 of 7 compared to the controls (Figures 2 and 3).

Figure 2

Case 8 of CVV on cell layers. (A) Original material, presence of scarce mycotic elements and inflammatory response; (B) control cell layer: there is a BF of Candida albicans; (C) CK activity on the cell layer of Candida albicans: only altered scarce blastospores are seen and there is absence of EPM.

Figure 3

Case 9 of CVV on cell layers. (A) Original material, presence of scarce mycotic elements and inflammatory response; (B) control cell layer: there is a BF of Candida albicans; (C) CK activity on cell layer of Candida albicans: only altered scarce blastospores are seen and there is absence of EPM.

DISCUSSION

The use of antibacterial and anti-mycotic combinations is very popular for local treatment of genital tract infections, whether by using gels, creams, or ovules and suppositories. They are an important option in the prophylaxis and treatment of superficial infections. In addition, they have low incidence of systemic toxicity and lower development of resistance than drugs of parenteral administration. It is known that 65% of human infections are associated with the formation of BF⁽⁷⁾. The genital tract is not different and we have showed, like other authors, that BF are formed in endogenous infections, such as bacterial vaginosis and CVV, and work as a real reservoir, therefore it is difficult to eliminate them and as such, they are usually associated with recurrent infections^(7,10,11).

The combinations used do not always comprise the activity on the BF. We have chosen two of those that use different antimicrobials: clindamycin associated with ketoconazole and that including metronidazole, miconazole, gentamicin, neomycin, polymyxin, and centella.

Clindamycin belongs to the group of lincosamides, together with lincomycin. It is a synthetic derivate of lincomycin that was obtained in 1966. Owing to its larger activity, lower absorption through the gastrointestinal pathway and the larger spectrum, the previous one was replaced in the clinical practice.It was first introduced as an anti-staphylococcus. It was later seen as a powerful anti-anaerobe. Although the risk of colitis through Clostridium difficile has limited its use, this is a useful antibiotic for treating severe infections by anaerobic microorganisms. Lincosamides are composed of an amino acid (methyl proline) and a sugar (pyranose) that are united by an amide. In the clindamycin, the hydroxyl is replaced in the 7th position by a chloride atom.

Even though clindamycin is bacteriostatic, its bactericide action has been seen against some strains of Staphylococcus, Streptococcus, and Bacteroides.

It also inhibits the bacterial protein synthesis by connecting to the 50S subunit of the bacterial ribosome, thus preventing the beginning of the peptide chain.

The location of connection in the ribosome is the same for macrolide and chloramphenicol, therefore they inhibit their actions by competence and should not be used together because they are antagonistic. When used in vitro, they inhibit the production of staphylococci toxins associated with the toxic shock syndrome and prevent the production of BF. By changing the surface molecules, clindamycin facilitates the opsonization, phagocytosis, and intracellular death of bacteria, even in sub-inhibitory concentrations. The consequent alteration of the bacterial wall decreases the adherence capacity of bacteria like of Staphylococcus aureus to host cells and facilitates their destruction.

It also has a long-lasting post-antibiotic effect against some susceptible bacteria, possibility owing to the persistence of the drug in the ribosome union place. Its immunomodulatory activity in vivo is notorious and it is capable of suppressing the synthesis of toxins in Streptococcus pyogenes. Therefore, it is usually associated with the classical treatments using penicillin in severe infections⁽¹²⁾.

Metronidazole is a molecule with anti-parasitic and anti-anaerobic activity that lacks immunomodulatory properties. The other antibacterial agents have different effects, but none of them works on BF or penetrate in its inner side.

The azoles that use both preparations have similar pharmacodyna-mics; however, similarly to all of them, they do not have the ability to penetrate through the EPM that form BF of Candida spp. They can work, but they do it slowly until they damage the mycotic elements.

The endogenous infections of the lower genital tract include the CVV, which affect a significant number of women at reproductive age. Topic and systemic medications are used for their treatment. The use of probiotics⁽¹³⁾ is also suggested, since they would difficult the formation of hyphae, which is the most virulent form of Candida spp.

The pathogenesis of CVV is a multifactorial process and Candida spp may interact among themselves and with other microorganisms⁽¹⁴⁾.

This is the reason for antimicrobial combinations. In order to eliminate the participating microorganisms, one has to obtain the penetration of the antimicrobial agents in the infection location and achieve proper concentrations⁽¹⁵⁾. The BF are usually resistant to many antifungal drugs⁽¹⁶⁾.

Our results show that both the species used in the formation of BF and also those from the patients that we studied with CVV, show an EPM that would make the arrival of anti-mycotic agents difficult. The combined use of CLI and KET improves the activity of KET. The CLI facilitates the dispersion of the EP from the BF, even though it does not adhere. Thus, KET may act more freely on Candida cells. This is confirmed with the isolated action of CLI on BF of CA and CAN.

It is clear that CLI does not have any action on yeasts, but it facilitates the action of KET and possibly of any other azole. When other antibacterial agents that do not disperse the EPM such as the CLI are used, the efficacy of anti-mycotic agents could be almost null. The activity would be done on the signal molecules, thus interfering with the EP structure that includes blastospores and hyphae. Some activity on the other EP components could be postulated. This would be better manifested in vivo since other microorganisms present in the genital tract could contribute to the formation of BF and to the structure of EP⁽¹⁷⁾.

CONCLUSION

According to these results, the use of CK in endogenous vaginal tract infections because of Candida spp is more useful than the ketoconazole alone, due to the activity of clindamycin on the EPM architecture that presents the Candida spp biofilm. It is usually present in these infections, especially in recurrent or complicated ones.