Antibiotic resistance profiling of Uropathogenic Enterobacteriaceae, Casablanca, Morocco

. Introduction: the urinary tract infection is a pathology frequents as well in community as at the hospital. In last years, there has been increased the incidence of antibiotic resistance in Uropathogenic Enterobacteriaceae. This development explains the need for periodic regional surveillance of this resistance to antibiotics, which poses a real problem of public health, because it affects several molecules ranging from β -lactam to quinolones. Objective: the aims of this study is to determine the profile of antibiotic resistance of Uropathogenic Enterobacteriaceae isolated from medical analysis laboratories in Casablanca, Morocco. Materials and methods: This is a prospective study that was conducted on patients who visited medical analysis laboratories in Casablanca city, for urinalysis test from 01 January 2017 to 30 December 2018 (a two full years). The culture was performed according to the usual techniques, and the antibiogram was done according to the recommendations of the Antibiogram Committee of the French Society of Microbiology. The statistical analysis was performed using Microsoft Excel ( Microsoft 2016 ). Results : During our study, we reported 18% of urinary tract infections, Sex ratio F/M was 1.79. In addition, Enterobacteriaceae were the most common germs. Of all the strains isolated, we noted a predominance of Escherichia coli with 75.41%) and Klebsiella ssp with 17.05%. In the course of this study period, we observed high rates of resistance to the main classes of antibiotics, and an overall increase between 2017 and 2018 in this resistance to the majority of β -lactam antibiotics, quinolones and aminoglycosides for almost all species of Uropathogenic Enterobacteriaceae. However, amikacin was the most active molecule against the Uropathogenic Enterobacteriaceae strains isolates. Conclusion: The development of resistance of Uropathogenic Enterobacteriaceae to antibiotics is a worrying phenomenon that exposes us to difficulties in the therapeutic management of infections. The current control of antimicrobial resistance is a real emergency and requires the involvement of public awareness before government instructions.


Introduction
Urinary tract infections (UTIs) are a severe public health problem and are caused by a range of pathogens, but most commonly by Enterobacteriaceae family: Escherichia coli, Klebsiella spp, Proteus spp, Enterobacter spp [1]. According to the world health organization "WHO", urinary Enterobacteriaceae infection is one of the most common infections acquired in community and hospital [2]. The routine therapy of UTIs is based on the use of antibiotics such as β-lactams, quinolones and aminoglycosides in many countries. Unfortunately, the widespread and misuse of these antibiotics resulted in the increasing rate of resistance to them in the societies [3]. In the last years, the increase in rates of antibiotic resistance among Enterobacteriaceae has posed challenges in choosing regimens, especially when infection due to multidrug resistant Enterobacteriaceae are suspected or endemic. Also, it's become a major international public health issue, raising fears of therapeutic impasses, that's why it has the priority for surveillance and resistance studies given their high frequency and severity [4,5]. In Morocco, the data on resistance to antibiotics of Uropathogenic Enterobacteriaceae are very limited and needs further investigations [6,7]. Therefore, the global purpose of our study is to describe the antibiotic resistance profiling of Uropathogenic Enterobacteriaceae strains isolated at medical analysis laboratories in Casablanca, Morocco, in order to supervise their emergence and to restrict the spread in the population.

Setting
This study was designed as a point-prevalence survey performed between 01 January 2017 and 31 December 2018, it was conducted in medical analysis laboratories, Casablanca, Morocco. Casablanca is the economical capital of Morocco and the largest city in country.
The activities of those laboratories consist in carrying out medical analyzes on behalf of private clients, clinics and hospitals, it admitted patient from all specialties and all ages from all regions of Morocco.

Study population and data Collection
2090 urines samples were collected throughout Casablanca from the health facilities, for each patient who came to the laboratory for a cytobacteriological examination of urines, a sterile sampling tube was given.
Patients were assisted with strict hygiene measures to ensure an aseptic sample. Prior to the collection, a survey from was made available to capture patient's details, these were related to personal and medical information.

Collection and identification of strains
The biologic criteria of inclusion were a pure bacterial culture with a colony count ≥ 105 CFU/ml associated with a leukocytes count ≥104/ml. Culture was performed by inoculating 10 mL of urine onto CLED (Cystine Lactose Electrolyt Deficien) agar. After incubation at 37°C aerobically overnight, bacteria were identified using routine microbiologic procedures including biochemical test (API 10E or 20E system-bioMerieux®, Marcy l'Etoile, France and Vitex 2 compact 15 system-bioMerieux®).

Antimicrobial drug susceptibility testing
The study of antibiotic susceptibility was carried out by the Muller Hinton agar (Bio-Rad, Marnes-la-Coquette, France) diffusion technique and VITEK 2 compact 15 system bioMerieux® cards. Following overnight incubation, the inhibition zone diameters were measured, each strain was classified as susceptible (S), intermediate (I) or resistant (R) with interpretative reading according to the recommendations of the antibiogram committee of the French Microbiology Society (CA-SFM) [8].

Statistical analysis
Data were analyzed using the statistical software Excel, Microsoft 2016. This data included the identification number, the patient's name, the date of isolation, the germ identified, as well as the antibiotics tested with their susceptibility profile (S, I, R).
Categorial variable were expressed as percentages, and continuous variable were expressed as means ± SD or median or median. Prevalence of infected patients were calculated.

Patient characteristics
During the study period, 2090 urine samples were collected, included 374 infected patients had urinary tract infection (18% of positivity).
The average age of patients infected in our series was 34.77 ± years old with extremes age ranging from 1 month to 97 years.
The most common symptoms observed among patients infected are fever and urinary and kidney problems. Urinary cytology showed erythrocytes, polynuclear neutrophils infiltrates, epithelial cells, yeasts and urinary crystals, of which calcium oxalate crystals were the most numerous.
The female sex was predominant with 64.17% female compared to 35.83% male, with a F/M sex ratio of 1.79 (240 females and 134 males patient).
There were 306 non-duplicate Enterobacteriaceae isolates recovered from the infected patients (some of these patients had UTIs caused by more than one organism in the same episode).
The majority of uropathogens were Escherichia coli with 72% (230 strains, followed by Klebsiella spp (17.05%). On the other hand, the other Uropathogenic species (Enterobacter spp, Proteus spp) were also isolated but with very low proportion. The distribution of the bacteria in patients with UTIs is shown in Figure 1

Antibiotic Resistance of Uropathogenic Enterobacteriaceae
In the course of our study, we sought to determine and interpret the profiling of Uropathogenic Enterobacteriaceae to various antibiotics tested according to CASFM standards.

Profile and development of β-lactams antibiotics resistance
The antibiotic resistance profiling of the different Uropathogenic germs shows a high resistance rate of E. coli for ampicillin (63.16%), Ticarcilin (57.87). As well as, for the amoxicillin-clavulanic acid combination (38.16%). The resistance rate of E. coli strains to 3rd generation cephalosporin was 16.9% and 16.96 % for Cefoxitin (2rd generation cephalosporin).
In addition, Enterobacter spp had significant resistance to β-lactams with percentages of 76.72%, 61.54% respectively for the combination of Amoxicillin-Clavulanic acid and 3rd generation cephalosporin.
We also noted that 77.77% of Proteus spp strains are resistant to the combination of Amoxicillin-Clavulanic acid. While cephalosporin remains the most active antibiotics on these strains. Figure 2       Rate of quinolones antibiotics resistance for Enterobacteriaceae summarized in Figure 5.

Conclusion
Uropathogenic Enterobacteriaceae constitute, in our city, an increasing infectious risk with high levels of resistance to antibiotics and the emergence of resistance to last resort molecules in antibacterial therapy, they can lead, in many cases to a therapeutic impasse.
This phenomenon of multi-resistance to antibiotics is a major public health problem in Morocco, worrying and alarming because of the potential risks (increased morbidity and mortality, additional economic costs and installation of highly resistant bacteria in hospital services and in the community).
Better control in terms of strict compliance with hygiene measures, isolation of carriers, the reasoned use of antibiotics and defining therapeutic and prophylactic strategies adapted to local epidemiology are the key actions to slow down their emergence and dissemination.
In addition, compliance with the rules of good pharmacy practice for the delivery of drugs, the major role that city pharmacists must play as advisers in antibiotic therapy are all elements to be promoted in controlling the spread of multi-resistance in the community.
The intra-and extra-hospital spread of multidrug-

Conflicts of Interest
Authors declare no conflict of interest.