اثرات سینرژیتیک ضد باکتریایی بتاکلروال-آلانین و فسفومایسین بر روی جدایه‏های ادراری اشرشیاکلی

Introduction

Urinary tract infections (UTIs) are the most common infectious diseases that occur in the community as well as healthcare setting and E.coli is the main agent of this disease (1). Antibiotic therapy is the gold standard for treatment of UTIs, however this microorganism is becoming resistant to the agents that are normally prescribed. This leads to several numbers of management and therapeutic problems. This fact has caused the need to develop new antimicrobial drugs (2- 4).

E.coli is a rod-shaped Gram-negative bacterium and a member of Enterobacteriaceae family.Different types of antibiotics are used for the treatment of UTIs caused by E.coli (5).The shortage of new antimicrobial agents has made the scientific community reconsider the potential value of old antibiotics.Phosphomycin is used for single-dose treatment of uncomplicated urinary tract infection due to E.coli especially in women (6, 7) but developing bacterial resistance to this antibiotic has limited its usage (8).Many studies showed high efficacy of phosphomycin in treatment of non complicated UTI caused by E.coli (9).Guidelines recommended three options for first-line treatment of acute uncomplicated cystitis; including phosphomycin, nitrofurantoin and cotrimoxazole (10), however long term therapies even with very effective antimicrobial drugs may result in numerous side effects and cause selection of resistant bacteria.

Phosphomycin interferes with the bacterial cell wall biosynthesis through the inhibition of UDP-N-Acetyl glucosamine 3 enolpyruvyl transferase (MurA) (11).MurA enzyme connects phosphoenol pyruvate (PEP) to the 3-OH group of UDP-N-Acetyl glucosamine that finally leads to the production of one of the peptidoglycan subunits termed Acetyl muramic acid.There are some other chemicals such as phosphinates, thiazolidines and β-Chloro-L-alanine that inhibit peptidoglycan biosynthesis (2, 3, 7, 12 & 13).β-Chloro-L-alanine is a non-toxic amino acid analog that inhibits Alanin-Valine transaminase or transaminase C (MurC) irreversibly.MurC is the enzyme responsible for the assembly of L-alanin to UDP-MurNAC during the intra-cytoplasmic stage of peptidoglycan biosynthesis (13).It seems that two peptidoglycan biosynthesis inhibiting agents have synergetic effect as they used in combination.The synergistic activities of phosphomycin with β -lactam antibiotics as well as β-Chloro-L-alanine with methicillin have been reported previously (14).Phosphomycin and β-Chloro-L-alanine both inhibit the intra-cytoplasmic stages of peptidoglycan biosynthesis and in respect to rapid occurrence of resistance to phosphomycin, its clinical usage becomes limited (15), in order to overcome to this disadvantage of phosphomycin, we tested the hypothesis that sub-MIC doses of Phosphomycin as a Mur A inhibitor antibiotic, may have synergistic effects in combination with β-Chloro-L-alanine as a safe Mur C inhibitor on UTIs isolates of E.coli.

Material and Method

Bacterial isolates and culture media

A total of 40 isolates were collected from urine specimens submitted to the clinical diagnostic laboratories in Urmia, Iran during a 12 months period from March 2010 to February 2011.The isolates were further processed by standard methods (16) to identify as E.coli.Isolated bacteria was maintained for long storage on skim milk medium (BBL; Becton Dickinson Microbiology Systems Cockeysville, MD21030, U.S.A). by adding 10% glycerol in -80°C.E.coli (ATCC25922) was used as the reference strain (17).

Determination of antibacterial activity of phosphomycin and β-Chloro-L-alanine

 The frozen bacterial isolates were inoculated on nutrient agar medium and then cultured overnight at 36±0.5°C.The bacteria were suspended in sterile buffer saline and used as inoculate within one hour after adjustment.Bacterial inoculate were added to serial dilutions of phosphomycin (Sigma-Aldrich) or β-Chloro-L-alanine (Sigma-Aldrich), with final bacterial concentration of 1.5×10⁶ cell /mL by adjusting with Mc Farland's Turbidity Standard No.0.5.MICs and MBCs of phosphomycin and β-Chloro-L-alanine against tested isolates were determined by dilution method using Mueller Hinton Broth (BBL) according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2006).The range concentration for determining MIC and MBC values for phosphomycin and β-Chloro-L-alanine was considered as 0.25-128 mg/Land 0.0625- 1 mM respectively.The experiments were carried out in triplicate.The MIC and MBC values were defined as the lowest antibiotic concentration that completely prevented turbidity in broth or colony growth on agar medium after incubation at 37°C for 24 hour respectively (18).

Determination of synergetic activity of phosphomycin with β-Chloro-L-alanine

Nutrient broth medium with sub-MIC concentrations of phosphomycin for each isolate was prepared, fixed concentrations of β-Chloro-L-alanine (0.5 mM) was added to each tube.1.5×10⁶ CFU/mL of each isolate were inoculated in each tube and incubated at 36±0.5°C.After overnight incubation, MIC and MBC of phosphomycin in the presence of 0.5 mM β-Chloro-L-alanine were determined (19).Synergy was described as if there was a reduction in MIC (or MBC) of phosphomycin in the presence of 0.5 mM of β-Chloro-L-alanine for each isolate (20).

Results

From 40 E.coli isolates, only 12.5% were susceptible to all investigated concentrations of phosphomycin and 2.5% were resistant, so in the selected range of phosphomycin concentrations, we could determine MIC and MBC values for 85% of isolates.The mean MIC value for phosphomycin in the other isolates was determined as 25.7± 35.5 mg /L, all the bacterial isolates were resistant to investigated concentrations of β-Chloro-L-alanine.In vitro application of β-Chloro-L-alanine and phosphomycin combination decreased the MIC as well as MBC values in 22.5% of the isolates (Fig.1 and 2).

Fig.1- Reduction of phosphomycin MBC in nine E.coli isolates.

As demonstrated in Fig.1, β-Chloro-L-alanine has significantly decreased the MBC values of phosphomycin for nine isolates.(Black: MBC of phosphomycin for each isolate, Gray: MBC of phosphomycin in the presence of 0.5 mM β-Chloro-L-alanine for each isolate).

Fig.2- Reduction of phosphomycin MIC in nine E.coli isolates.