ANTIBIOTIC SUSCEPTIBILITY ANALYSIS OF CLINICAL BACTERIAL ISOLATES IN CAIRO, EGYPT

Hussien H. El-Sheikh 1 ,SoheirAbdelRahmanSalih 2 , Mahmoud M. Elaasser 3 , Nesreen A. Safwat 3 and Mostafa Y. Ibrahim 1 . 1. Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Cairo, Egypt. 2. Department of Clinical pathology, Faculty of Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt. 3. The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In the present report, among one thousand and six hundred urine samples screened; only 65% showed bacterial infection. However, 919 samples (88.4%) were sensitive to the tested antibiotics; 121 samples (11.6%) recognized as multi-drug resistant bacteria. These bacterial isolates were identified and differentiated by cultural, morphological, and biochemical analysis. The isolates were then analyzed to determine their susceptibility profile to 30 antibiotics according to the standard CLSI guide.The results also indicated that the most of urinary tract infection diseases were by Gram negative bacteria (102 isolates; 84.3 %). Escherichia coli was the most predominant organism causing UTI in this study that represented by 58 isolates (47.9%), followed by Klebsiella pneumonia (21.5%), Pseudomonas aeruginosa (14.9%), Staphylococcus aureus(12.4%), Enterococcus faecalis (3.3 %).In conclusion, the profiles in bacterial clinical isolates can provide important information for the control of antibiotic resistance as well as distribution and susceptibility profiles in populations.
It has been estimated that more than 60% of healthcare-associated infections are biofilm-related. Urinary tract infections (UTIs) are the most common type of hospital-acquired infection, accounting for 40% of all cases and of those 80% are catheter-associated (Hamill, 2007).
The incidence of urinary tract infections is much higher in adult women due to anatomical and physiological reasons. However due to the urinary tract lesion formation in males and children, it is more dangerous and must be treated immediately to suppress the infection and prevent spreading. A wide spectrum of treatment can be ranging from a single-dose antibiotic treatment of simple cystitis in young females, to rescue nephrectomy for pyonephrosis in diabetic patients with septic shock (Gupta et (Theodros, 2010;Abdagireet al., 2014).
In the current study, the susceptibility pattern of pathogenic bacteria isolated from Egyptian UTI patients with special reference to multi-drug resistant bacteria. The development of microbial resistance to the available antibiotics has informed the need to explore natural disease control options.

Materials and Methods:-
Samples Sources and collection:-Clinical specimens were obtained during a period of 24 months (from September 2011 to August 2013). Urine samples were collected from 1600 patient's (males, and females) of different ages from in patients and out patients of two hospitals: Al-Zahraa University Hospital and Cairo Specialized Hospital in Cairo city, Egypt. Every patient got a sterile dry, wide opening and leak proof container. A "mid-stream" or "clean-catch" urine sample obtained under aseptic precautions in sterile containers. In babies, the urine is collected in sterile self-adhesive plastic bags (Simervilleet al., 2005). Some patients (e.g., elderly people or hospitalized patients) that cannot provide a urine sample in such cases, a catheter was inserted into the bladder to collect urine, this is the best method for providing a contaminant free sample. Specimens were transported in sterile containers and analyzed within one hour of collection (Mahdyet al., 2012).

Laboratory Investigations for Urine Samples:-
Urine specimens were analyzed by dipstick and microscopy, and then cultured. Test strips (Medi-Test Combi 10 Oxoid ® , UK) were performed for rapid determination of blood, urobilinogen, bilirubin, protein, nitrite, ketones, ascorbic acid, glucose, pH value and density in urine. Then wet film of urine deposit is examined for pus, RBCs, casts and crystals. The white blood cells (pus cells) are counted. Also, bacterial count is done within one hour by using a calibrated loop that carries a 10µl of uncentrifuged urine. A loop-full is spread on blood agar, and on MacConkey or on cystine lactose electrolyte deficient (CLED) agar (Oxoid ® , UK) and incubated overnight at 37 o C. The number of bacteria is estimated and interpreted as follows:  1x10 5 bacteria per ml or more indicates urinary tract infections.  Less than 1x10 4 bacteria per ml are regarded as contamination.  A count of 1x10 4 is considered significant if the organism is Gram positive and of one type only.

Isolation and Purification of Uropathogenic Bacteria:-
Patient's specimens were taken from urinary tract infection cultured by the "Plating out" technique, on solid media. The media used for isolation of bacteria (aerobic as well as facultative anaerobic bacteria) from urine samples under investigation were nutrient agar medium, MacConkey agar medium, Blood agar medium, Chocolate agar medium and Cysteine-Lactose-Electrolyte-Deficient (CLED) medium purchased from Oxoid, UK. All plates were incubated aerobically at 37 o C for 24 hours. The colonies were picked up by sterile loop and subjected to purification in the same isolation medium. Agar streak method was used for purification process. A well separated colony from each isolate was picked up on isolation medium, slants and incubated at 37 o C for 24 hrs. The purified isolates were subjected to a complete identification process and other studies.

Methods of Identification of Bacterial Isolates:-Morphological Studies:
The morphological characteristics of the purified bacterial isolates were carried out according to the identification charts (Holt et al., 1996).
Examination of unstained preparations (wet mount) will help in demonstrating motility. While examination of Gram stained preparation-will determine the staining reaction of the organism; whether Gram-positive or Gram-negative, their morphology (cocci, bacilli, etc.), size and arrangement (Chessbrough, 2000).

Physiological and Biochemical Identification:-
Many biochemical reactions were preceded for identification of bacteria according to the keys of Bergey's manual of determinative bacteriology (Hensyl, 1994).

Identification of The Bacterial Isolates by Analytical Profile Index (API) strips:-
The identification of all bacterial isolates was performed using API strips inoculated and incubated as described by the manufacturer (bio Merieux ® France). The strips were API 10S, API 10 Strep and API 20 staph. Examination of the strips was conducted after 18-24 hr. incubation at 37°C. The analyses were done using API instrument (bio Merieux ® Vitek Systems).

Antibiotic Sensitivity Tests:-
Antibiotic sensitivity of uropathogen isolates were preformedin-vitro by modified Kirby-Bauer disk diffusion method using Mueller-Hinton agar medium (Oxoid ® , UK), according to the recommended method of Clinical Laboratory Standards Institute (CLSI, 2012). The Mueller-Hinton Agar plates were inoculated with the bacterial suspension within 15 minutes after adjusting the turbidity of the inoculum suspension, by dipping a sterile cotton swab into the suspension. The swab was then streaked over the entire surface of the Mueller-Hinton medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum. The disc diffusion method was applied using commercial paper discs impregnated with antibiotics (Oxoid ® , UK). 30 different antibiotic discs were applied to the plates within 15 minutes after inoculation. By using a multidisc dispenser, the appropriate discs were placed on the surface of the inoculated plate suitably spaced (25 mm from disc to disc and 15 mm from the rim). The plates were allowed to pre-diffuse at 4 o C for one hour. Plates were incubated at 37 o C for 24 h., and then measured the inhibition zones diameter in millimeter (mm) including the disc (6mm). According to the diameter of the inhibition zone, as documented from standard antimicrobial sensitivity charts for the different antibiotics, it can be determined if the organism is sensitive, intermediate or resistant to the different antibiotics according to Clinical Laboratory Standard Institutes guidelines (CLSI, 2012).

Results and Discussion:-
As the result of increasing mis-use and extensive uses of antimicrobial agents, nosocomial pathogens have shifted away from easily treatable bacteria towards more resistant bacteria. This change is important problem for nosocomial infection control and prevention (Jain et al;2007).
In this study during 2011 to 2013, urine samples were collected in Al-Zahraa University Hospital and Cairo Specialized Hospital in Cairo city from 1600 patient's males and females of different ages. The results showed that among the 1600 urine samples collected from patients 1040 samples exhibited bacterial growth (65%) and 560 samples (35%) did not showed any bacterial growth under these screening conditions (Fig.1). Among the 1040 bacterial-infected patients, only 121 samples (11.6%) recognized as multi-drug resistant bacteria. However, 919 samples (88.4%) were sensitive to the tested antibiotics.Among the ward samples collected; the frequency of multidrug resistant patient are higher (75.2%) in In-patient samples (n= 91). Moreover, the out-patient samples (n= 30) represent only 24.8% (Fig. 2). From the screening results, it was found that the adult females are the most patients infected by urinary tract infections (n=69, 57.0%), followed by adult males (n=31, 25.6%), then child females infected (n=13, 10.8%) more than child males (n=8, 6.6%) as shown in fig. (3).For adult females infected with urinary tract infection, the percentage of non-pregnant women about (95.7%, n=66) and the remaining were pregnant women (4.3%, n=3) (Fig. 4).Regarding to sample type, the majority of samples infected with bacteria were fresh mid-stream urine (n=115, 95.04%), while the urine samples from urinary catheter (n=6) representing 4.96% (Fig. 5 However, all bacterial isolates were screened concerning their sensitivity to different antibiotics on Mueller Hinton agar mediumby disc agar diffusion method.The total pattern of resistance according to each antibiotic of the different 30 antibiotics used is represented for selection of antibiotic resistant bacteria, 121 bacterial isolates were called multi resistant bacteria after compare the inhibition zone around each antibiotic disc with inhibition zone standard according to (NCCLS, 2014).     The most efficient antibiotic used in this study was Imipenem (IPM); hence 89.3% of tested clinical bacterial isolates were sensitive to this antibiotic; 6.6% were intermediate and 4.1%were resistant.  Table 1).
1496    Significant difference was observed between genders as majority of the pathogens were isolated from females. Studies conducted all over the world have reported the differences in the prevalence rates between females and males due to physiological and anatomical differences (Theodros, 2010;ColganandWilliams, 2011;Hooton, 2012).
UTIs are considerably more common among women, nearly half of whom will experience a UTI during their lifetime (Schollum and Walker, 2012).It is estimated that 2 to 10% of pregnant woman suffer from any form of UTIs (Sharamiet al., 2007; Lee et al., 2008;Bahadiet al., 2010), which compatible with the obtained results in this study.
Pregnancy appears to increase the risk that a UTI because pregnancy can cause hormonal changes, as well as shifts in the position of the urinary tract, which make it easier for bacteria to spread to the kidneys (Hooton, 2012).
In this study, the antimicrobial susceptibility tests revealed that one hundred and twenty-one (11.6%) isolates had multidrug resistance. Also, in the current study, the survey of antibiotic susceptibility revealed that Imipenem was the most effective drugs, inhibiting 89.3% of the isolated bacterial uropathogens. The other three most sensitive antibiotics exhibited susceptibility rates 71.1%, 57%, 43% were Polymyxin B,Meropenem and Amikacin, respectively.This has been observed previously in a study involving 211 clinical strains (Thibaultet al., 2004) and is of interest because Imipenem antibiotic is considered as a good alternative to ceftazidime in the treatment of disseminated disease.
Ahsanet al., (2011) reported that the highest resistance rate of Escherichia coli isolate which was obtained from urine samples was against nalidixic acid (57.7%) followed by cotrimoxazole, ciprofloxacin, and ampicillin, respectively. However, in our study amikacin and imipenem had the widest coverage against Escherichia coli isolates (97.5%). In another surveillance study reported that the highest resistance rate of Escherichia coli isolates which were obtained from various hospitals specimens was against tetracycline followed by amoxicillin and penicillin, respectively (Mohammadtaheriet al., 2010; Kang et al., 2011).
However, in the current study Escherichia coli, the most frequently isolated bacterium, showed high resistance rates (>80%) to erythromycin, amoxycillin and tetracycline. Similarly, Klebsiellaspp., Proteus spp.and Pseudomonas spp. were found to be resistant to amoxycillin, erythromycin and tetracycline but sensitive to gentamicin and ciprofloxacin. Increasing drug resistance to these and other antimicrobials has been documented from previous studies (Tseng et al., 2008).
This is in concordance with the prevalence rate reported from Gupta et al.(2002) though resistance against gentamicin (50.4%), ciprofloxacin (38.8%) and amikacin (20.6%) was much higher than seen in our study.
Results obtained from Ghorashiet al. (2011), suggested that Escherichia coli was extremely resistant to ampicillin but highly sensitive to amikacin in most of the hospitalized patients.
According to Ebenebeet al., (2014),Pseudomonas showed 100% resistance to all the beta-lactam antibiotics except Imipenem. All the Gram-positive bacteria were susceptible to Imipenem. National and regional distributions of the data areimportant to enable local prescribing practices (Laureet al., 2012). The carbapenem (imipenem) with activity against many bacteria havebeen the most active broad-spectrum antimicrobialclass documented by numerous large surveillanceprograms (Zhanelet al., 2007).

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The high levels of antimicrobial resistance inGram-negative bacteria can be attributed to antibioticmisuse in Egypt (Ashour and El-Sharif, 2009).
More likely, Atif (2006) and Jumaa (2006)  In conclusion, policies on the control of antibiotic usage have to be enforced and implemented to avoid the evolution of newer generations of pathogens with higher resistance, not only to the older generation drugs, but also to the relatively new ones. In addition, the entire microbial spectrum in various infection sites, and not just bloodstream pathogens, should be taken into account when initiating empirical antibiotic therapy.