INTRODUCTION:

Pseudomonas aeruginosa is a non-fermentative, aerobic, Gram-negative rod shape microorganism that can occupy the soil and aquatic environments with minimal nourishing needs and high adaptability to numerous ecological unfavorable conditions. It is usually isolated from clinical specimens of hospitalized patients primarily those with suppressed immune defenses and counted as one of the topmost widely disseminated nosocomial and life-threatening health pathogens^1,2,3. Pseudomonas aeruginosa is ranked one of the main causatives of plenty of diseases, for example, ventilator associated pneumonia, surgical site, and urinary tract infections^4,5. It represents about 12 % of all hospital acquired infections and rated as the first worldwide pathogen causing burn-wound infections which in a lot of cases progress to sepsis resulting in high incidence of burn-wound associated mortality. In addition, Pseudomonas aeruginosa was observed to be the second topmost cause of pneumonia and the fourth topmost cause of urinary tract infections (UTIs) globally^,6,7. Worryingly, Pseudomonas aeruginosa infections are highly problematic to be cured due to its natural resistance and the momentous capacity to challenge conventional antibiotic groups⁸. In addition, it has rapid evolutionary tactics to acquire adaptive resistance towards different antibiotics, bringing about the upgrowth of multi-drug resistant (MDR) strains. MDR Pseudomonas aeruginosa isolates is considered an earnest problem and their infections have turned into a huge threat in numerous countries around the world with limited options of treatment^9,10,11. Infections caused by resistant isolates are an issue of great concern as they are accompanied by 3-fold higher mortality rates and 9-fold higher rate of secondary bacteremia¹². Since the antibiotic susceptibility profile of Pseudomonas aeruginosa is in continuous change, this study, like many other studies on susceptibility profile, aimed at determining the most proper empirical antimicrobial therapy that can be helpful in the efficacious management of pseudomonal infections. Likewise, these studies will also help in limiting the expansion of antimicrobials resistance that occurs due to the inappropriate use of antibiotics^13,14.

MATERIALS AND METHODS:

Collection of specimens:

Clinical specimens from different sources (urine, sputum and wound-burn infections) were taken from patients who admitted to Port Said General Hospital, Port Said, Egypt during the period from May 2016 to December 2016 according to standard procedures^15,16. Briefly, sputum specimens were taken by using sterile wide-mouth containers with a secure, tight-fitting cover and requesting patients to cough deeply to produce sputum. Urine specimens were taken from clean-catch midstream urine in sterile, wide-mouth containers. Burn-wound specimens were taken using sterile cotton swabs from the middle of the burn-wound site after swabbing around the burn-wound site with a sterile alcohol swab and then the cotton swabs are added to sterile tryptone soy broth (Oxoid, UK). The specimens were transported and processed immediately at the laboratory of Microbiology and Immunology Department- Faculty of Pharmacy- Port Said University where the study was conducted.

Isolates identification:

Pseudomonas aeruginosa isolates in this study were identified following conventional methods¹⁷. Specimens were streaked on nutrient agar (Oxoid, UK) and incubated at 37°C under aerobic conditions for 24 hr. Each colony isolated from nutrient agar was initially examined by Gram-staining. Colonies which appeared as Gram-negative rods were further sub-cultured on MacConkey agar (Oxoid, UK). Pale colonies on MacConkey agar which exhibit non-lactose fermenter isolates were tested by oxidase test, citrate utilization, growth and pigment production on cetrimide agar (Oxoid, UK) and the capability to grow at 42°C which is characteristic for Pseudomonas aeruginosa.

Antibiotic discs used:

All Pseudomonas aeruginosa isolates in this study were tested against standard anti-pseudomonal antibiotic discs including, aztreonam 30 µg (ATM), piperacillin 100 µg (PRL), ceftazidime 30 µg (CAZ), cefepime 30 µg (FEP), ciprofloxacin 5 µg (CIP), levofloxacin 5 µg (LEV), amikacin 30 µg (AK), gentamicin 10 µg (CN), colistin sulfate 10 µg (CT) and imipenem 10 µg (IPM). The anti-pseudomonal discs were procured from (Oxoid, UK).

Antimicrobial susceptibility testing:

The antimicrobial susceptibility testing for the isolates was implemented using the disc diffusion technique as stated by the Clinical Laboratory and Standards Institute (CLSI) guidelines¹⁸ against the above mentioned anti-pseudomonal antibiotics. Briefly, well-grown colonies of tested isolate were suspended in 5 ml sterile saline solution and adjusted to match the turbidity of 0.5 MacFarland. This solution was swabbed over the surface of Muller Hinton agar(Oxoid, UK) plates. The plates were swabbed in three directions (the plate was rotated 60º) to ensure equal distribution, and then the anti-pseudomonal discs were placed on the plates surfaces. The plates were incubated aerobically at 37ºC within 30 minutes of preparation for 18 hr. After incubation, the diameter of the inhibition zones was measured and recorded in mm and the results were interpreted according to the diameters in (CLSI)¹⁸, Figure 1.

Figure (1): Representative examples of antimicrobial susceptibility test of some tested Pseudomonas aeruginosa isolates.

RESULTS:

Pseudomonas aeruginosa isolates and identification:

A total of 150 positive Pseudomonas aeruginosa isolates were recovered from the patients. All the 150 Pseudomonas aeruginosa isolates were confirmed phenotypically as they all were Gram-negative rods, non-fermenters on MacConkey agar, gave positive oxidase test and positive citrate utilization test and exhibited an ability to grow on cetrimide agar with green pigmentation at 42°C. The distribution of Pseudomonas aeruginosa isolates among different types of specimens is presented in Table 1.

Table (1): Distribution of Pseudomonas aeruginosa isolates among different types of specimens

Specimen type

Urine

Sputum

Burn-wound

Total No. (%) of all isolates

Number and percentage of isolates

(29.33%)

(40.67%)

(30%)

150

(100%)

Antimicrobial susceptibility and resistance profile:

According to the type of specimens, it was found that the 44 Pseudomonas aeruginosa isolates from urine specimens showed the highest susceptibility against colistin sulfate 39/44 (88.64%), followed by imipenem with susceptibility equal to 33/44 (75%), while the highest resistance of urine isolates was against piperacillin 10/ 44 (22.73%), Table 2. On the other hand, the 61 Pseudomonas aeruginosa isolates from sputum specimens exhibited the highest susceptibility also to colistin sulfate 60/61 (98.36%), followed by both aztreonam and ciprofloxacin with susceptibility equal to 49/61 (80.33%), while the highest resistance of sputum isolates was against gentamicin 20/61 (32.79%) and both piperacillin and ceftazidime 14/61 (22.95%), Table 3. Finally, the 45 Pseudomonas aeruginosa isolates from burn-wound specimens showed the highest susceptibility to colistin sulfate 37/45 (82.22%) followed by levofloxacin with susceptibility equal to 34/45 (75.56%), while the highest resistance of burn-wound specimens was obtained against gentamicin 19/45 (42.22%) and both piperacillin and ceftazidime 17/45 (37.78%), Table 4. Moreover, the resistance behavior of Pseudomonas aeruginosa isolates in this study showed that 45 of the isolates (30%) are MDR (resistant to one antimicrobial agent in at least three different antibiotic groups), Table (5). The antibiotics which showed the highest resistance or the highest sensitivity against the tested isolates are bolded in the tables below.

Table (2): Antimicrobial susceptibility pattern of Pseudomonas aeruginosa isolates from urine specimens.

Susceptibility pattern Antibiotics tested	No. (%) of resistant isolates (R)	No. (%) of intermediate isolates (I)	No. (%) of sensitive isolates (S)
ATM	6 (13.64%)	6 (13.64%)	32 (72.72%)
PRL	10 (22.73%)	11 (25%)	23 52.27%)
CAZ	4 (9.09%)	11 (25%)	29 (65.91%)
FEP	3 (6.82%)	11 (25%)	30 (68.18%)
CIP	5 (11.36%)	9 (20.46%)	30 (68.18%)
LEV	1 (2.27%)	13 (29.55%)	30 (68.18%)
AK	4 (9.09%)	14 (31.28%)	26 (59.09%)
CN	5 (11.36%)	16 (36.36%)	23 (52.27%)
CT	5 (11.36%)	0 (0%)	39 (88.46%)
IPM	8 (18.18%)	3 (6.82%)	33 (75%)

Table (3): Antimicrobial susceptibility pattern of Pseudomonas aeruginosa isolates from sputum specimens.

Susceptibility pattern Antibiotics tested	No. (%) of resistant isolates (R)	No. (%) of intermediate isolates (I)	No. (%) of sensitive isolates (S)
ATM	4 (6.56%)	8 (13.11%)	49 (80.33%)
PRL	14 (22.95%)	11 (18.03%)	36 (59.02 %)
CAZ	14 (22.95%)	1 (1.64%)	46 (75.41%)
FEP	12 (19.76%)	4 (6.56%)	45 (73.77%)
CIP	9 (14.75%)	3 (4.92%)	49 (80.33%)
LEV	12 (19.67%)	1 (1.64%)	48 (78.69%)
AK	12 (19.67%)	6 (9.84%)	43 (70.49%)
CN	20 (32.79%)	1 (1.64%)	40 (65.57%)
CT	1 (1.64%)	0 (0%)	60 (98.36%)
IPM	9 (14.75%)	4 (6.56%)	48 (78.69%)

Table (4): Antimicrobial susceptibility pattern of Pseudomonas aeruginosa isolates from burn- wound specimens.

Susceptibility pattern Antibiotics tested	No. (%) of resistant isolates (R)	No. (%) of intermediate isolates (I)	No. (%) of sensitive isolates (S)
ATM	8 (17.78%)	7 (15.56%)	30 (66.67%)
PRL	17 (37.78%)	4 (8.89%)	24 (53.33%)
CAZ	17 (37.78%)	3 (6.67%)	25 (55.56%)
FEP	15 (33.33%)	3 (6.67%)	27 (60%)
CIP	5 (11.11%)	7 (15.56%)	33 (73.33%)
LEV	11 (24.44%)	0 (0%)	34 (75.56%)
AK	13 (28.89%)	6 (13.33%)	26 (57.78%)
CN	19 (42.22%)	5 (11.11%)	21 (46.67%)
CT	8 (17.78%)	0 (0%)	37 (82.22%)
IPM	13 (28.89%)	2 (4.44%)	30 (66.67%)

Table (5): Resistance pattern of MDR Pseudomonas aeruginosa isolates.

Isolate No.	Number and names of agents in which each isolate resist	Isolate No.	Number and names of agents in which each isolate resist
P1	4 (CAZ, FEP, CN and IPM)	P24	6 (CAZ, FEP, CIP, LEV, AK and CN)
P2	5 (PRL, CAZ, FEP, AK and CT)	P25	4 (ATM, PRL, CAZ and FEP)
P3	7 (PRL, CAZ, FEP, CIP, LEV, AK and CN)	P26	8 (PRL, FEP, CIP, LEV, AK, CN, CT and IPM)
P4	7 (ATM, PRL, CAZ, FEP, AK, CN and IPM)	P 27	5 (CAZ, FEP, LEV, CN and IPM)
P5	7 (ATM, PRL, CAZ, FEP, AK, CN and IPM)	P28	8 (ATM, PRL, FEP, CIP, LEV, AK, CN and IPM)
P6	4 (LEV, AK, CN and IPM)	P29	6 (ATM, PRL, CAZ, FEP, AK and CN)
P7	3 (LEV, CN and CT)	P30	6 (PRL, CAZ, FEP AK, CN and IPM)
P8	7 (ATM, PRL, CAZ, FEP, AK, CN and IPM)	P31	9 (ATM, PRL, CAZ, FEP, CIP, LEV, AK, CN and IPM)
P9	5 (FEP, LEV, CIP, AK and CN)	P32	5 (CAZ, FEP, LEV, CN and IPM)
P10	3 (LEV, CN and CT)	P33	6 (ATM, PRL, CAZ, FEP, AK and CN)
P11	5 (CAZ, FEP, CIP, LEV and CN)	P34	8 (ATM, PRL, FEP, CIP, LEV, AK, CN and IPM)
P12	7 (CAZ, FEP, CIP, LEV, AK, CN and IPM)	P35	7 (ATM, PRL, CAZ, FEP, AK, CN and IPM)
P 13	4 (ATM, PRL, AK and CN)	P36	6 (ATM, PRL, CAZ, FEP, AK and CN)
P14	6 (PRL, CAZ, FEP, AK, CN and IPM)	P37	5 (CAZ, FEP, LEV, CN and IPM)
P15	8 (CAZ, FEP, CIP, LEV, AK, CN, CT and IPM)	P38	7 (ATM, PRL, CAZ, FEP, AK, CN and IPM)
P16	4 (PRL, CAZ, FEP and IPM)	P39	6 (ATM, PRL, CAZ, FEP, AK and CN)
P17	6 (PRL, FEP, CIP, LEV, AK and IPM)	P40	6 (CAZ, FEP, CIP, LEV, AK and CN)
P18	3 (PRL, CAZ and CT)	P41	3 (PRL, CN and IPM)
P19	3 (PRL, CAZ and CT)	P42	8 (ATM, PRL, CAZ, FEP, AK, CN, CT and IPM)
P20	3 (PRL, LEV and AK)	P43	6 (ATM, PRL, CAZ, FEP, CN and IPM)
P21	3 (PRL, CAZ and CN)	P44	6 (ATM, PRL, CAZ, FEP, CN and IPM)
P22	6 (PRL, CAZ, FEP LEV, CN and IPM)	P45	4 (LEV, AK, CN and CT)
P23	5 (ATM, CIP, LEV, CN and IPM)

DISCUSSION:

Regardless of the advances achieved in the last decades in the medical care and infection control protocols, Pseudomonas aeruginosa still considered a leading cause of serious human infections which can be frequently isolated from hospitalized patients^3,19,20. In the current study, Pseudomonas aeruginosa was isolated from different patients experiencing UTIs, pneumonia and burn-wound injuries. The distribution of Pseudomonas aeruginosa isolates in different studies may vary according to the geographical regions and the hospital from which the specimens were collected because each hospital and health facility has a different environment associated with it⁶. Increasing antimicrobial resistance has become a major threat around the world as it decreases the effectiveness of antibiotic treatment of microbial disease^21,22. The rising resistance rate of Pseudomonas aeruginosa to various anti-pseudomonal agents has been reported worldwide which is a critical therapeutic problem in the handling of infections due to this microorganism^6,23. Antimicrobial susceptibility test is conclusive in clinical practice and remains the ideal approach to nominate the most suitable antibiotics for treating Pseudomonas aeruginosa infections in order to control it and decrease the mortality rates¹². In our study, Pseudomonas aeruginosa isolates from urine specimens exhibited the highest susceptibility against colistin sulfate (88.64%) and imipenem (75%) while isolates from sputum specimens were highly sensitive to colistin sulfate (98.36%) and to both aztreonam and ciprofloxacin (80.33%). In addition, isolates from burn-wound specimens were highly susceptible to colistin sulfate (82.22%) and levofloxacin (75.56%). Similar results, showing that colistin and quinolones are the drugs of choices for treatment of pseudomonal infections, were reported in several studies performed worldwide. For instance, in a study, it was reported that colistin was the most effective antibiotic against Pseudomonas aeruginosa isolates from urine (93.3%) which is close to our results²⁴. In another study, colistin also showed the highest susceptibility towards Pseudomonas aeruginosa urine isolates (100%) which is in accordance with the present study²⁵. In a similar study, colistin and levofloxacin showed the highest susceptibility of (90.9%) and (60.2%) respectively against Pseudomonas aeruginosa isolates which is close enough to our results²⁶. Numerous factors participated in the spread of MDR isolates in Egypt primarily the increased disaster of antibiotic misuse and biocides²⁷. In the current study, the resistance rate of the isolates from urine was the highest to piperacillin (22.73%), while the isolates from sputum were highly resistant to gentamicin (37.79%) and both piperacillin and ceftazidime (22.95%) and the isolates from burn-wound were also highly resistant to gentamicin (42.22%) and both piperacillin and ceftazidime (37.78%). Earlier studies in Egypt and globally also reported that Pseudomonas aeruginosa isolates were highly resistant to gentamicin, piperacillin and 3^rd generation cephalosporins including ceftazidime^26,28,29. The MDR rate in this study was 30% which matches the rates reported previously in Egypt which ranged from 30%-56%^28,30,31. Other global studies exhibited lower MDR rates; 5.9% in Canada³² and 19% in Germany³³. This elevated MDR rate in Egypt in comparison to other countries gives us an alarm to the necessity of the application of rigorous antibiotic prescription strategies. In conclusion, although the high speed of resistance evolving among Pseudomonas aeruginosa and the presence of considerable percentage of MDR strains, Pseudomonas aeruginosa isolates in this study were susceptible to colistin sulfate and with lower degree to the quinolones (ciprofloxacin and levofloxacin) which is a good information for the prescribers in Egypt to give careful consideration in their future rational prescriptions and use of these antibiotics should be the main focus. Although colistin sulfate produced the highest susceptibility results, we suggest the usage of quinolones as the first choice because quinolones have much lower side effects in comparison to colistin sulfate.

CONFLICT Of INTEREST:

The authors declare that they have no conflict of interest.

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Received on 05.05.2018 Modified on 31.05.2018

Research J. Pharm. and Tech 2018; 11(8): 3268-3272.

DOI: 10.5958/0974-360X.2018.00601.7