Antimicrobial Activity of Non-bond Colicin on Candida albicans Biofilm

Two hundred fifty mid-stream urine specimens were collected from Baqubah Teaching Hospital and Al-Batool Teaching Hospital from patients with urinary tract infections (UTI). Of these investigated urine specimens, 66 (26.4%) specimens showed positive growth culture of Gram-negative bacteria. From these, Escherichia coli was the most prevalent bacteria of the examined culture (41, 62.12%). Additionally, the cup assay was used to determine colicin producers while the most efficient colicin producers were estimated by the formation of larger inhibition zone. Approximately half of the investigated E. coli isolates (20, 49 %) was colicin producers. Colicins was extracted after induction by mitomycin-C showed a concentration of 3020 μg/ml, as estimated utilizing the Lowry method, while its activity was 80 U/ml. Our study results showed that colicin had significant antibiofilm activity (P≤ 0.05) against Candida albicans and the effect seemed to be concentration dependent. . However, the values of biofilm inhibition varied depending on the different tested isolates. The biofilm of isolate 5 showed the most significant inhibition (P≤ 0.05) by colicin with a value of 46%, while isolate 3 was less affected with an inhibition rate of 19% at the concentration of 2500 μl/ml.


Introduction
Escherichia coli is one of the most common pathogens, which causes a wide spectrum of diseases within and outside the intestinal tract]1[. Extraintestinal pathogenic E. coli (ExPEC) is the main causative agent of urinary tract infection, enteritis, septicemia and other infections such as neonatal meningitis ]2[. One of its key pathogenicity features is the production of bacteriocins]3[. Bacteriocins are ribosomal synthesized antimicrobial peptides that have the ability to kill or inhibit the growth of other strains, without damaging the producing bacteria due to having specific immunity proteins ]4[. These peptides are different in many features like molecular mass, the existence of post-translational modifications, mechanisms of bacteriocins release from producer cells, and others ]5[. Genes of bacteriocin biosynthesis are clustered and encoded on plasmids, chromosome and/or transposons ]6[. It is believed that the killing mechanism of colicins produced by E. coli can be accomplished by pore formation in the inner membrane of the target cell and degradation of intracellular components such as DNA and RNA ]7[. Candida albicans is a dimorphic fungus that may be found as commensal in the oral cavity of healthy people, but it also causes recurrent, severe and even lethal systemic infections ]8[. It is thought that the rising rate of immunocompromised patients could lead to increase the risk of candidiasis ]9[. C. albicans can infect skin, mouth, throat and blood ]10[. This may be attributed to the possession of many virulence factors that help C. albicans to infect the host. C. albicans virulence factors include polymorphism, adhesins and invasions, hydrolases, germ tube formation and biofilm ]11[. Biofilm is a population of microorganisms attached to the solid surfaces and embedded in extracellular polymeric substances (EPS) that are composed of proteins, carbohydrates, and nucleic acids. Microorganisms usually produce these EPS matrix in a complex structure, which is comparable to honeycombs of the hive, that supports them as a mechanical defense and resistance against antimicrobials ]12[. Patients can acquire infection due to the presence of biofilms on hospital equipment and medical devices, eventually leading to persistent infections ]13[. There are several reasons to analyze the effect of colicin as antimicrobial against candida, such as the appearance of antimicrobial resistance candida and the side effects of these drugs as well as the spread of drug resistant biofilms.

Materials and methods Isolation and identification of bacterial and fungal isolates
Mid-stream urine specimens were collected from Baqubah Teaching Hospital and Al-Batool Teaching Hospital from patients clinically diagnosed with urinary tract infections (UTI). The isolates were identified utilizing microscopic examination. Morphological features of the colonies and biochemical tests were conducted according to Brenner and Farmer]14[ as well as by using chrome agar and Vitek-2 system. C. albicans isolates were obtained from oral swabs from patients with renal impairment. C. albicans isolates detection was confirmed by forming germ tubes ]15[ and by Vitek-2 system.

Detection of colicin-producing isolates
Colicin-producing E. coli were detected using cup assay]16[. The most efficient producers showed the largest inhibition zone and the feature of the stability of bacteriocin production.

Extraction of crude non-bound colicin.
Previously incubated 2.5ml of nutrient broth with the selected colicin producer were added to sterile nutrient broth supplied with 5% of glycerol and then incubated for 14h at 37 ℃. After the addition of 2μg/ml of mitomycin-C, they were incubated in an incubator shaker for 3 hrs then centrifuged at 5000 rpm for 30 min using refrigerated centrifuge. The non-bound colicin in the supernatant was separated from the cells. To eradicate the remaining cells, chloroform was added. To confirm the bacterial clearance, the supernatant was cultured on brain heart infusion. The activity of colicin was detected by using the well method ]17[ and colicin concentration was estimated by Lowry method ]18,19[.

Biofilm formation of C. albicans
After incubation of C. albicans isolates on sabouraud dextrose broth, they were diluted by sterile broth at the ratio of 1:20. Each well of the 96-well flat microtiter plates were filled with 200 μl of these isolate suspensions. Sabouraud dextrose broth was also used as negative control in separate wells of the 96-well flat microtiter plate. Experiments were performed in triplicates in which the plates were incubated for 48hrs at 37 ℃. Following the incubation, the medium and the unbound cells were  isolates among other G-bacteria urine specimens isolated from UTI patients.
In this respect, a previous study reported that the prevalence of E. coli (38.90%) was higher than the other bacteria in UTI cases ]24[. In this regard, several conditions may affect the prevalence of bacteria among patients. These may involve environmental, health, social, and cultural conditions of patients. In addition, the technical mistakes for isolation and identification of bacteria may give inconsistency in the reported findings]25[.

Detection of colicin-producing E. coli by cup assay
By the detection of the inhibition zone using cup assay, we observed that 20 E. coli isolates (49%) were colicin producers (Figure-3) and the most effective isolate had the larger inhibition zone.

Biofilm formation of C. albicans
Biofilm formation of C.albicans was assessed by the microtiter plate method [20]. The isolates 2 and 5 were found to form strong biofilms and their optical density values were 0.36 and 0.337, respectively, while moderate biofilms were found to be formed by the others isolates, with optical density ranged from 0.249 for isolate 1 to 0.313 for isolate 7 ( Table-1).

Inhibition of biofilm formation by non-bond colicin
The results of biofilm inhibition were dependent on the concentration of colicin and the type of indicator E. coli isolate. This was evident when the higher concentrations of colicin led to significantly (P≤ 0.05)increased inhibition of biofilm; . Thus, the relation between the extracted colicin concentration and the biofilm inhibition is inversely proportional in the tested C. albicans isolates .
Colicin producers E.
coli 49% Non-colicin producers E. coli 51% The results demonstrated in Table-2 show that all C. albicans isolates were inhibited significantly (P≤ 0.05) by colicin, but their sensitivity was variant depending on the isolates. The isolates 1 and 5 were the most sensitive to colicin at the concentration 2500 µg/ml, with inhibition of biofilm values of 43% and 46%, respectively. At the same concentration, isolate 3 was the less sensitive isolate that showed a value of biofilm inhibition of 19%. There are some explanations of colicin action on the biofilm of microorganisms. Some bacteriocins act by disrupting the co-aggregation process of the membranes which is important for biofilm stability; thus it decreases biofilm development by reducing its biomass and thickness ]32[.
Other bacteriocins cause pore formation that results in an efflux of ATP from biofilm cells. The size of pores has to be larger than 1.5 in diameters which is enough to cause efflux of ATP ]33[. Moreover , some bacteriocins have the ability to suppress biofilm genes such as atl (autolysin) and ica (intercellular adhesin) such as bacteriocin gallidermin ]34[. Furthermore, ColA-43862 produced by Citrobacter freundii is known to have anti-biofilm activity, but it may not act as a limiting factor due to the complication of biofilm and its microenvironment that act as a barrier of colicins action ]35[. An earlier study reported that a bacteriocin of Lactobacillus acidophilus had remarkably reduced biofilm cells of catheter-associated multidrug-resistance Pseudomonas aeruginosa. This bacteriocin can act as an alternative for antibiotics that hardly eliminate biofilm of P. aeruginosa ]36[. A recent study reported that a bacteriocin of Bacillus subtilis (subtilocin) caused biofilm inhibition of Gardnerella vaginalis, with an inhibition value higher than 90%, however, it did not decrease the growth of planktonic cells. Also, it remarkably inhibited the biofilm of E. coli and L. monocytogenes. Inhibition of biofilm by these bacteria occurs because of their ability to inhibit the quorum sensing (QS) ]37[. Bacteriocin EntV of Enterococcus faecalis has a reduction activity on virulence factors of C. albicans without affecting the viability of cells. It blocks hypha formation, which results in preventing biofilm formation as well as reducing inflammation and invasion of the epithelium by candida in marine models ]38[.

Conclusions
Biofilm of C. albicans was significantly inhibited (P≤ 0.05) by crude non-bond colicin and the inhibition effect was more evident at high concentrations of the extracted colicin. However, the biofilm inhibition effect of the E. coli extracted colicin seemed to be C. albicans isolates-specific as some isolates were more sensitive to colicin while others were less affected.