Antibiosis resistant of Pseudomonas aeruginosa isolated from different clinical specimens

Objective: Detection of antibiotic sensitivity of Pseudomonas aeruginosa isolated from different clinical specimens in Kirkuk, Iraq. Material and Methods: The present study included (3138) samples collected from different clinical specimens from outpatients and inpatients of both sexes with different ages who were attended Azadi Teaching Hospital/ Kirkuk during the period from October/2007 until May/ 2013. Results: From a total of 3138 Specimens, 1485 gave positive cultured and 1653 Specimens showed no bacterial growth, 319/3138 (10.17%) isolates were identified as P. aeruginosa. These isolates were identified according to morphological, cultural and biochemical characteristics. Wound and ear swab were important source for P. aeruginosa and isolated more frequently in inpatients than outpatients. The rate of isolation in females 169/319 (52.97%) was higher than males 150/319 (47.01%). Antibiotic susceptibility test of these isolates was performed, and the results showed that all Pseudomonas isolates (100%) were resistant to ampicillin, cephradin and trimoxazole, followed by gentamycin (97.3%), Amoxicillin (97.3), cephalexin (92.3%), neomycin (91.4%), nalidixic acid (89%), nitrofurantoin (87.5%), tobramycin (87.5%) and ciprofloxacin (84%), and the resistance to amikacin was (75%). Antibiosis resistant of Pseudomonas aeruginosa isolated from different clinical specimens Siham Sh. AL-Salihi 1 , Braihan H. Hameed 2 , Braihan H. Hameed 3


INTRODUCTION
Pseudomonas aeruginosa (P. aeruginosa) is an aerobic, motile, gram negative rod bacterium that belongs to the family pseudomonadaceae, [1] usually found in soil, water, plants, animals and humans [2] and is commonly present in moist environments in hospitals.
It can colonize normal humans, in whom it is a saprophyte [3]. Pseudomonas aeruginosa is one of the most frequent and dangerous pathogens involved in the etiology of severe nosocomial infections [4]. It has been implicated in diverse nosocomial infections like nosocomial pneumonias, urinary tract infections (UTIs), skin and soft tissue infections, in severe burns and in infections in immunocompromised individuals [5]. Infections caused by P. aeruginosa are often life threatening and difficult to treat because of its primary limited susceptibility to commonly used antimicrobial agents [6]. Most strains of P. aeruginosa are multidrug resistant [7]. The development of bacterial resistance is a major worldwide problem complicating the use of chemotherapeutic agents and the control of infectious diseases [8].
Thus, the aims of the present study were to isolate and identify of P. aeruginosa from outpatients and inpatients from different clinical specimens (blood, urine, wound and ear infections) and assess of the antibiogram profile of the isolates.

Sample collection
The study was conducted in the Medical Microbiology Laboratory of the Azadi Teaching Hospital/Kirkuk between October/2007 to May/2012. Approximately (3138) samples were collected from different clinical specimens (blood, urine, swabs from wounds and ear infections) from outpatients and inpatients of both sexes with different ages.

2.2Bacterial isolation and identification
Collected samples were cultured onto nutrient, blood and MacConkey's agar for primary isolation. Non-lactose fermented colonies were selected and cultured onto Cetrimide Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com 19 agar, then incubated overnight at 37°C for refreshment and demonstration of their ability for blood hemolysis [9, 3].

Antibiotic sensitivity (disc diffusion test)
McFarland standard solution was used in antimicrobial susceptibility test, tube No. 37°C for 18 to 24 hours. After incubation, zone of inhibition was recorded in millimeter using metric ruler [11,12] and translated into predetermined categories as resistant and sensitive [13].

Isolation and identification of P.aeruginosa
In the current study, all bacterial isolates were cultured selectively using cetrimide medium and according to microscopic characteristics, cultural and biochemical tests (Table.1). These colonies were identified as P. aeruginosa. The selected colonies were tested for oxidase and catalase production and sub-cultured on MacConkey's agar to obtain pure culture for further diagnosis investigations. All the isolates that produced pyocyanin (blue pigment), and are able to grow at 42°C but not at 4°C, these criteria were depended in our study for identification of P. aeruginosa from other species not possess these criteria which  20 neglected and do not included in the study results [3]. Under microscope it appears as gram negative, rod-shape, and occurs as single, pairs, or in short chains [14].

Alkaline slant, Alkaline butt, No H2S and No gas production
In this study, the prevalence of P. aeruginosa isolates in clinical specimens examined over the study period was (10.17%) ( Table.2), this level is relatively low when compared with similar studies with higher prevalence level. In India, Savaş et al. reported a level of (20.3%) [15], while (30%) was reported in a study conducted in Pakistan [16], and (18.25) in Egypt [17]. However, this finding is higher than a study in northeastern Nigeria which found that a level of (2.1%). Comparison of epidemiological data of bacterial pathogens as in this study might be difficult as there are other variables that influence the outcome of results such as, clinical specimens received for examination, studied population, type of hospitals and geographical locations [15].  In the (Table.3), the rate of isolation in female 169/319 (52.97%) was higher than male 150/319 (47.01%), the reason may be that female more infected with urinary tract infection and exposed to burning and wounded during cooking more than male. A study reported that P. aeruginosa is responsible for (12%) of hospital acquired urinary tract infections, (8%) of surgical wound infections, and (10%) of bacteremia [18].
In this study, the distribution of isolates also differs with other studies, a study done by Latif in Iraq who showed that P. aeruginosa were most common (44.4%) in burn infection, followed by (38.1%) ear infection, (16.6%) wound and (6.6%) urinary tract infection, while P.
aeruginosa cannot be isolated from eye infections [19].
There are differences in the percentage of infections between our results and others, and the reasons for these variations in all studies may be due to the percentage of distribution of isolates which varied according to the place of clinical samples collection, environmental factors, nutrition requirements and virulence factors [24].

Antibiotic susceptibility testing
The present study showed that all wound samples isolates were resistant nearly to all 12 tested antibiotics ( (87.5%) and ciprofloxacin (84%), and the resistance to amikacin was (75%). A major problem in P. aeruginosa infection is the resistance to relatively high levels of most antibiotics in use [25]. It has been emphasized that there is a remarkable increase in the incidence of infection by antibiotic resistance microorganisms in different parts of the world. A study indicated that P. aeruginosa infections are difficult to treat as this organism displays a high level of intrinsic antibiotic resistance [26]. Until the end of the 1980s, fluoroquinolones had excellent activities against P.
aeruginosa but extensive use of the antimicrobial, in particular ciprofloxacin, had led to an increasing incidence of ciprofloxacin-resistant isolates [27]. Whereas at 1984 almost (100%) of P. aeruginosa isolated in the USA, Europe and Japan were susceptible to ciprofloxacin [28]. The current study showed that ciprofloxacin resistance was (84%). This finding is higher than that in a study in India which found that the resistant of ciprofloxacin against P.
aeruginosa was (73.2%) [29]. ISSN 1992 -0849 Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com 24 The current study revealed that antipseudomonasal effect of amikacin is higher than gentamycin. This finding correlates with other studies conducted by Smitha et al and Poole et al who found that resistance to amikacin of P. aeruginosa was still lower than to gentamicin [30,31]. Other study reported that P. aeruginosa showed higher levels of resistance to gentamicin and imipenem [32].
Another study found a group of P. aeruginosa strains lacked β-lactamase activity but were resistant to penicillins, ceftazidime, ciprofloxacin and amikacin which is due to impermeability or multidrug efflux (intrinsic mechanism of resistance) [33].
The present study is in agreement with a study which found that P. aeruginosa isolated from patients demonstrated resistance to ampicillin, gentamycin and nalidixic acid [34].
However, disagrees with other study which observed that all P. aeruginosa isolates were resistant to ampicillin, cephalexin and nalidixic acid while they were sensitive to ciprofloxacin and tobramycin [19].
Antibiotic resistance is now generally accepted as a major public health issue. P.
aeruginosa infection is considered a major problem because of its resistance to relatively high levels of most antibiotics in use [35], particularly due to the combination of the following mechanisms: betalactamase production, a strong barrier to diffusion at the outer bacterial membrane and bacterial efflux. Selective pressure of antimicrobial drugs has an important impact on the development of bacterial resistance [36].