Antimicrobial effects of amniotic membrane on some bacterial strains

1Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Microbiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 3Department of Microbiology, Fasa University of Medical Sciences, Fasa, Iran 4Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran Immunopathologia Persa http www.immunopathol.comm


Introduction
Bacterial keratitis is a menace and concern in the optical aspects and considering antibiotic resistance development in bacterial agents, substitution or combination therapy may be essential (1).If untreated, cornea infection may develop towards perforation and consequently glaucoma, cataract and endophthalmitis may occur as the outcomes (2,3).The most common agents are Pseudomonas aeruginosa, Escherichia coli, Group A streptococci and Staphylococcus aureus.P. aeruginosa is a very common (up to 70%) agent in the cornea infection (1,4,5).Infection and production of degrading enzymes by these species play a critical role in the invasion and destroying cornea cells (6).On the other hand, bacterial cell wall compounds

Key point
The amniotic membrane has antimicrobial properties and can be used alongside antibiotics for the treatment purposes.
interact with Toll-like receptors and initiate an inflammatory response via production of cytokines and chemokine molecules.The subsequent inflammatory response induces the cornea tissue damage.Furthermore, polymorphonuclear leukocytes cells (PMNs) migration and release of lysosome and oxidative compounds play an important role in the bacterial eradication.These responses may cause cornea tissue damage as well (7).Severe bacterial keratitis needs rapid and extended use of local antibiotic therapy.However, these compounds are toxic for the epithelial cells and have potential of harm to these cells after a period of consumption.The antibiotic resistance has made many difficulties for treatment of these infections.The human amniotic membrane (AM) is the inner layer of placenta and is composed of three layers including cuboidal epithelium, thin stem membrane and stromal matrix (8).Several studies have shown that both AM and liquid exhibit the antimicrobial and wound healing properties.Surveys have revealed the presence of natural antimicrobial peptides including alafin, SLPI and 1-3HBD in the epithelium of the AM (9)(10)(11)(12).The antimicrobial effects of AM have been demonstrated against several species such as E. coli, Group A streptococci, P. aeruginosa and S. aureus (13)(14)(15)(16).AM graft for epithelial reformation has been employed in order to eradicate the P. aeruginosa infection of keratitis and have exhibited desirable outcomes (17).

Objectives
The aim of the current study was to assess antimicrobial properties of AM against P. aeruginosa keratitis in vivo and over some other standard strains in vitro.

Bacterial strains
Five strains including P. aeruginosa ATCC27853 (from Shiraz University of Medical Sciences, causing keratitis), S. enterica BAA-708, E. coli ATCC25922, K. pneumonia ATCC7881, E. faecalis ATCC29212 (from Tehran University of Medical Sciences) were prepared for antimicrobial testing.The half McFarland of these strains was prepared.

Human amniotic membrane cutting and susceptibility testing
The human AM cuts were prepared in fresh from (with and without cocktails) the organ transplant, Imam Khomeini hospital bank in Tehran.The specimens were from placenta of women experiencing cesarean and negative for HIV, HBV, HCV and syphilis agents.Under sterile conditions, washing was done with normal saline and in next step with cocktails of cloxacillin 50 ug/mL, streptomycin 50 ug/ mL and amphotericin B 2/5 ug/mL.The specimens with and without antibiotics containing an equal volume of ice were transmitted to the health department of Medical Microbiology of Tehran University of Medical Sciences.For the antimicrobial susceptibility testing, the cut sizes were 1.5×1.5 and were placed on center of Muller-Hinton agar after bacterial culture similar to susceptibility testing protocol.A Para film was used as control negative and for ensuring the sterility, a medium without bacterial culture was used.After 24-hour incubation, the diameter of four directions was measured and the mean was obtained.

Environmental factors effects
The effects of temperature, pH and time on the antimicrobial effect of AM were assessed.In case of time effects, the diameters of growth inhibition were measured after 24, 48 and 72 hours of incubation.For pH effect, the susceptibility was evaluated in media with three different pH including 6.5, 7 and 7.5.Furthermore, for temperature effect, the plates were incubated in 25°C, 33°C and 37°C.

Animal model study of amniotic membrane effect
Eighteen polish rabbits with 1.5-2 kg were prepared from Razi Institute of Karaj, Iran.After one-week isolation, the rabbits were anesthetized and 0.5 cc of half McFarland of P. aeruginosa was injected into the stromal cornea using a 30G syringe attached to a micro syringe and by utilizing a biomicroscope to make keratitis after 20 hours.For membrane graft, after keratitis was observed, the rabbits were divided in two amniotic membrane transplantation (AMT) and control groups.The AMT group was anesthetized again and the AM was bound to the keratitis eyes using nylon sutures in direction of epithelium side following the sterile conditions by an optometrist (18,19).

Microscopic image capture and observation of results
Clinical observations of Pseudomonas keratitis and also corneal turbidity, epithelial impairs, descemetocele (protrusion of Descemet's membrane through the cornea), infiltration in stromal area and image capture before and after the membrane graft were measured in first, third and seventh days.The ulcer diameter of infiltration (due to the epithelial cells secretions) in stromal cornea which is the diagnostic criteria of effects of AM in this study was measured using ImageJ (version 1/44).The mean measurements of the numbers were obtained in each day of measuring.Moreover, the infiltration was compared between the AMT and the control groups.

Ethical issues
The research followed the tenets of the Declaration of Helsinki.Consent for operation and study had been taken.The ethical committee of Tehran University of Medical Sciences approved the research.All patients' information remained confidential.This study has the Iranian registry of clinical trials of IRCT 2015248N9.

Data analysis
The data were analyzed using SPSS version 16.Repeated measure analysis variance test was applied for comparison of means across one or more variables.P values less than 0.05 were considered as a significant result.

Amniotic membrane antimicrobial effects
The inhibition zone was measured for P. aeruginosa, S. enterica and E. coli strains, but not observed in case of E. faecalis and K. pneumonia and even the colonies were observed under the membrane (Figures 1 and 2; Table 1).

Effects of environmental factors
The results of temperature, bacterial concentration, time and pH variations effects on the inhibitory effect of AM showed no significant difference for all 5 strains when alterations fulfilled.

Animal model keratitis inhibitory effects
The corneal keratitis results were observed in both groups before membrane graft and 20 hours after injection of P. aeruginosa, including tear secretions, photophobia and conjunctiva hyperemia without microscope employment.The biologic microscopic observations included cornea inflammation, stromal ring infiltration and corneal turbidity (Table 2 and Figur 3).

Discussion
In the present study, the antimicrobial effect of AM was shown against three strains including P. aeruginosa ATCC27853, S. enterica BAA-708, E. coli ATCC25922, but K. pneumonia ATCC7881, E. faecalis ATCC29212 showed resistance to it.In the study by Kjaergaard et al, similarly the amniotic and carrion membranes exhibited inhibitory effect against group A and B streptococci, S. aureus, S. saprophyticus and E. faecalis in vitro, but in contrast their study demonstrated a narrow effect against E. coli and P. aeruginosa (13,14).Similar to the present study, in another study by Chakraborty and Bhattacharyya, the human amniotic extract exhibited a great inhibitory effect on E. coli DH5α (15).In the current study, similar to other previous data, the cesarean AM was used because of lower load of contamination.It has been noted that AM contains other components such as lysozyme, transferrin, immunoglubolin7S, β1 and β1a globulins and several others that can inhibit bacterial growth, and thus it should be carefully washed with sterile normal saline (20).It has been proposed that AM is a reservoir of antimicrobials transferring them to the cornea in a period of time without harm to the epithelium.In this study, the increase in inhibition zone in three susceptible strains using antibiotic cocktail is capable of diffusing the antibiotic from the surface with antibiotic to the surface without it (21,22).Almajano et al showed that the environmental factors and conditions interfered with the antimicrobial effects of AM.The pH effect on polyphenol antimicrobial properties led to the control of food contamination (23).However, in this study unlikely the pH showed no increasing effect on AM effects against susceptible strains.The alterations of minimum inhibitory and minimum bactericidal The inhibition zone was observed among all 5 strains by using membrane with antibiotic cocktail.(24,25).In this study, the incubation of plates in 25°C and 33°C instead of 37°C showed an increased activity against P. aeruginosa.In this study, the time duration of incubation showed no increasing effect on the inhibitory properties of AM.
Similarly, Talmi et al showed that AM incubation time in 24°C, 48°C and 72°C exhibited no different inhibitory results.The similar effect of AM in different environmental conditions suggests that related antimicrobial peptides are persistent in these conditions.In this study two bacterial dilutions including 1.5×10 6 and 1.5×10 7 demonstrated no significant difference in the inhibitory zone in any of strains.Similarly in the study by Talmi, use of 3×10 6 and 3×10 8 dilutions of group A streptococci exhibited no difference in inhibitory zone.
In the animal model study, AM graft could significantly decrease the keratitis signs.It has been shown that AM can remove bacterial effects on burn patients and is ideal burn wound dressing (26).Tehrani et al found that the preserved AM is a suitable substitute for the fresh AM in clinical situations (27).Obviously, Parthasarathy et al highlighted the presence of a potential antimicrobial activity of both amniotic and chorionic membranes to inhibit several bacterial and fungal pathogens in which maximum activity was recorded by AM (28).

Conclusion
The findings of this study showed that AM is an effective factor against bacterial strains in vitro and also demonstrated keratitis healing by decreasing the signs of descemetocele and perforation of corneal tissue.

Table 1 .
The growth inhibitory zones against bacterial strains

Table 2 .
Three groups tested in this study concentrations (MIC and MBC, respectively) in different temperatures have been shown in case of methicillin and penicillin-resistant S. aureus and E. faecalis