Antibiotic resistance among Escherichia coli urinary isolates and their susceptibility to clove essential oil

Escherichia coli is a Gram-negative, facultatively anaerobic, rod-shaped, coliform bacterium, which is a primary cause of urinary tract infections. Resistance to antibiotics has become a particular problem in recent decades. Consequently, there is an unmet need for new therapeutic options. It has been observed that essential oils have bactericidal effects. The antimicrobial susceptibility testing for Escherichia coli isolates obtained from urine of patients with urinary tract infections was determined via disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2015). Essential oil from clove – Syzygium aromaticum (L.) Merill et L.M. (Myrtaceae) was analyzed by GC-FID-MS. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were detected by using the micro-dilution broth method. Escherichia coli clinical isolates are characterized by high resistance to ampicillin, amoxicillin with clavulanic acid, norloxacin, trimethoprim/sulfamethoxazole, tetracycline, tobramycin and ticarcillin. Clove oil possesses strong inhibiting and killing properties against E. coli isolates, among them the ones resistant to recommended antibiotics. The results of this study highlight the need for testing the eficacy of new agents to inactivate bacteria in clinical settings.


INTRODUCTION
Urinary tract infection (UTI) is one of the most common conditions observed in general practice, accounting for up to 6% of consultations. About 150 million people worldwide develop UTI each year (9). It affects more women than men (24). 20% of women at any time have asymptomatic bacteriuria and around 40% of them develop at least one UTI during their lifetime (19).
Urinary tract infection (UTI) is the presence of urinary tract microorganisms above the bladder -generally > 10 5 / ml (signiicant bacteriuria). Asymptomatic bacteriuria means that bacteria tend to multiply up to 10 5 colony-forming units (CFU) per ml without any clinical symptoms (17). Symptomatic UTIs are classiied as cystitis, pyelonephritis, and urosepsis, considering that the urosepsis syndrome is the most severe form and that pyelonephritis is more severe than cystitis (26). zacché and Giarenis classiied UTIs as either uncomplicated or complicated depending on the presence of structural or neurological urinary tract abnormalities (28). The risk factors are phenotyped according to the ORENUC system: O indicates unknown risk factors; R -risk of recurrent UTIs but without risk of a more severe outcome; E -extraurogenital risk factors; N -relevant nephropathic diseases; U -urologic resolvable (transient) risk factors; C -permanent external urinary catheter and unresolved urologic risk factors (14).
Most UTIs are caused by the ascent of microorganisms through the urethra, although some microorganisms can reach the urinary tract by hematogenous or lymphatic spread (11). Most UTIs are monomicrobial. The most common uropathogen in uncomplicated upper and lower urinary tract infections is Escherichia coli (70-95% of cases) and Staphylococcus saprophyticus (5-10% of cases) (4,26). Occasionally there are isolated bacteria from Enterobacteriaceae family, such as Proteus mirabilis and Klebsiella spp. (20). The microbial spectrum of complicated UTIs is broader and includes species of Pseudomonas, Enterococcus, Staphylococcus, Serratia, and Providencia, viruses and fungi (21).
Escherichia coli is a Gram-negative, facultatively anaerobic, rod-shaped, coliform bacteria found in the lower intestine (25). Most E. coli strains are harmless, but some serotypes can cause serious disease (6). The harmless strains are part of the normal lora of the gut, can beneit their hosts Bacterial infections of urinary tract are treated with antibiotics or chemotherapeutics, at the initial empirical treatment period, and then on the basis of antibiogram. Polish recommendations for treating UTI include amoxicillin (and other semisynthetic penicillins), cephalosporins (mostly II and III generation), carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciproloxacin, nitrofurantoin and the aminoglycosides. According to the World health Organization, antibiotic resistance is a growing problem which poses a serious threat to public health. The consequences of infection with resistant microorganisms can be severe: increased mortality, prolonged stays in hospital, loss of prophylaxis for patients undergoing surgery and other medical procedures, as well as increased costs of treatment (5). Therefore, new therapeutic options should be proposed. This proves the need to develop new therapeutic options.
Strong antiseptic activity of essential oils has been known for many centuries. A large number of essential oils and their constituents have been investigated for their microbial properties against some bacteria. Clove oil is an essential oil extracted from the clove plant. There are three types of clove oil: bud oil derived from the lower-buds of S. aromaticum contains 60-90% of eugenol, leaf oil derived from the leaves contains 82-88% of eugenol, and stem oil derived from the twigs contains 90-95% of this compound (18). The antimicrobial activities of clove was proved against several bacteria and fungi which were the case of food contamination (27). Pure clove oil or mixes with rosemary oil were tested against E. coli and other bacteria. The results showed the strong growth inhibiting activity in concentration between 0.062% and 0.500% (v/v) (10).
The aim of this study was to investigate the patterns of resistance among E. coli isolates obtained from urine of patients with UTIs and check their susceptibility to clove essential oil.

MATERIALS AND METhODS
Escherichia coli clinical isolates were obtained from urine of patients with UTIs. The examination of urine samples was based on the presence of ≥ 10 5 CFU of microorganisms per ml in urine culture. The bacteria were cultured according to standard microbiological methods with the use of Columbia Agar (Graso, Poland) and Mac Conkey Agar (Graso, Poland). They were identiied using API 20 E tests (bioMerieux, France). Identiication was conirmed by using the automated instrument VITEK 2 (bioMerieux, France).
The antimicrobial susceptibility testing for Escherichia coli isolates was determined via disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2015). The following discs were used: AM -ampicillin (10 µg The composition of the clove oil was found to meet the requirements of the European Pharmacopoeia 8 (7) and of the Polish Pharmacopoeia IX (22) for the two main components: eugenol amounted to 86.0% (required 75.0-88.0%) and β-caryophyllene to 9.8% (required 5.0-14.0).

RESULTS AND DISCUSSION
The eficacy of antibiotic treatment depends on the identiication and determination of the uropathogen pattern of resistance. In our study 96.7% of UPEC isolates showed resistance to ampicillin, 73.3% to amoxicillin with clavulanic acid, 86.7% to ciproloxacin, 53.3% to norloxacine and 56.7% to trimethoprim with sulfamethoxazole. It means that the tested E. coli isolates were characterized by high resistance to penicillin and other antibiotics recommended in UTI. UPEC isolates also showed high resistance to tetracycline (70.0%) and tobramycin (46.7%). Among the cephalosporins, the most effective was ceftazidime (3.3% resistance), then: cefotaxim (6.7%), cefoxitin (13.3%) cefuroxime (16.7%), cefepime (16.7%) and cefalexine (20.0%). 13.3% of E. coli isolates were resistant to nitrofurantoine. A number of sensitive and resistant strains of E. coli are presented in Table 1. The tree diagram, shown in Figure 1, groups the applied antibiotics in terms of their effect on the tested strains.
The cluster analysis shows that among the used antibiotics 3 groups can be distinguished: the irst group includes antibiotics to which, most of E. The effectiveness of clove oil against E. coli isolates suggests that it could potentially be used as a supplement to UTI therapy or as an alternative therapy -all tested isolates were sensitive to studied oil. Based on preliminary studies, it was found that the MICs and MBCs for clove oil fall into the following range: MIC from 2.1 to 3.1 mg/ml and MBC from 3.1 to 4.2 mg/ml (Fig. 2). For more than 70% of clinical strains of E. coli, MIC and MBC values were 2.6 and 3.7 mg/ml respectively (Tab. 2).   (23) showed that the clove essential oil has a broad spectrum of antibacterial activity against Gram-negative bacteria. In this study clove oil inhibited the growth of all tested clinical isolates obtained from a range of clinical samples of stool, urine, blood and pus from wound. It was active against 100 isolates belonging to 10 different species of Gram-negative bacilli: Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella ozaenae, Klebsiella pneumoniae, Serratia marcescens, Salmonella typhi, Shigella dysenteriae and Vibrio cholera. The inhibition zones were between 11.87 ± 3.22 for E. coli isolates and 23.75 ± 3.03 for V. cholera isolates, but no data on the composition of the tested commercial clove oil. Fagere and Al Magboul (8) investigations conirmed the very strong antibacterial properties of clove oil against Gram-negative and Gram-positive bacteria. According to their study conducted with the use of the cup plate agar diffusion method and the microdilution method, the MIC of the clove oil against reference strains of Escherichia coli and Pseudomonas aeruginosa was 1.5 mg/ml, and the MBC against E. coli was 3.125 mg/ml and against P. aeruginosa was 6.25 mg/ml. We obtained the similar results of the MIC and MBC values in relation to the E. coli coming from urine, including those against isolates resistant to antibiotics recommended in UTI. Our tested clove essential oil was consisistent with the requirements for medical use. Other authors reported on the antibacterial and antifungal activity of clove essential oil also in combination with antimicrobial drugs and its potential in the treatment of urogenital infections (2). The clove essential oil is safe for humans at low concentrations. The World health Organization (WhO) established that the acceptable daily intake of clove oil per day is 2.5 mg/kg of weight in humans (16). The toxic effect has been proven for eugenol (as the main constituent), which is easily absorbed when administered orally, reaching rapidly plasma and blood with A cumulative effect has been hypothesized and associated to relieve of neuropathic pain after repeated daily administrations (12).
In conclusion, due to the fact that antibiotic resistant bacteria are a serious threat to public health worldwide, there is a need to search for new methods of controlling pathogens. A promising alternative to antibiotics are essential oils. Clove oil could be encapsulated and used as a supplement to conventional antibiotic treatment. The results of this study highlight the importance of testing the eficacy of new approaches to inactivate bacteria in clinically relevant settings.