pseudomonas aeruginosa is an important gram - negative opportunistic pathogen that has the capability to create variable clinical infections , including , but not limited to , wound infections , urinary tract infections ( utis ) , and blood stream infections ( bsi ) , particularly in hospitalized and immunocompromised patients ( 1 , 2 ) .
pseudomonas aeruginosa was the second most common cause of hospital - acquired pneumonia , as well as the third and fifth common cause of hospital - acquired utis in usa and europe , respectively ( 2 - 4 ) . evidently , p. aeruginosa due to low permeability of its outer membrane in combination with efflux pump overexpression is intrinsically resistant to a variety of antibiotics ( 5 ) .
expression of different classes of extended - spectrum -lactamas enzymes along with aminoglycoside - modifying enzymes ( aminoglycoside phosphoryltransferases , aminoglycoside acetyltransferases , and aminoglycoside nucleotidyltransferases ) , and mutation are other well - characterized mechanisms for antibiotic resistance in p. aeruginosa ( 6 , 7 ) .
data extracted from several independent studies in iran showed that p. aeruginosa was responsible for 11%-32.4% of utis , 17.2%-32.4% of pneumonia , 36.7% of bsi , and 47% of wound infections ( 8 - 12 ) . moreover , based on previous studies performed in iran , treatment of p. aeruginosa infections is a major concern for health - care setting due to its high resistant rate to different antibiotics ( 12 , 13 ) .
since determining bacterial isolates relatedness is essential for understanding the transmission routes , different typing methods that can be divided into two major categories , phenotypic and genotypic methods , have been established ( 14 ) .
phenotypic methods such as serotyping , pyocin typing , and antimicrobial susceptibility typing depend on the environmental factors , therefore , have low discriminatory power ( 14 , 15 ) .
genotypic methods have been performed by different typing techniques such as restriction fragment length polymorphism ( rflp ) , pulsed field gel electrophoresis ( pfge ) , multilocus sequence typing ( mlst ) , enterobacterial repetitive intergenic consensus - pcr ( eric - pcr ) , variable number tandem repeat ( vntr ) , dna hybridization and random amplified polymorphic dna ( rapd ) ( 16 - 21 ) .
although mlst and pfge have more discriminatory power , they are expensive , especially in developing countries ( 22 ) .
rapd - pcr has the advantage of being fast , reproducible , simple , and low - cost , thus , it is an appropriate approach for primary screening of epidemic strains in large number of isolates ( 23 , 24 ) .
to our knowledge , limited data are available about genetic diversity of p. aeruginosa recovered from different clinical specimens in isfahan ; the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran .
this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period
, a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .
primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .
the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) .
resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) .
the following disks ( mast , uk ) were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) .
p. aeruginosa standard strain ( atcc 27853 ) was used as the quality control .  
optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .
dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .
electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .
we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .
separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .
rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,
dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands
we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) .
this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period
, a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .
primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .
the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) .
resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) . the following disks ( mast , uk )
were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) .
optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .
dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .
electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .
we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .
separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .
rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,
dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands
we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) .
out of 150 samples , 54 ( 36% ) were positive for p. aeruginosa culture .
distribution of the 54 isolates were as follow , urine 39 ( 72.2 % ) , sputum 7 ( 13% ) , wound 5 ( 9.3% ) , blood 2 ( 3.7% ) , and eye discharge 1 ( 1.9% ) .
the most frequent involved wards were icu 28 ( 51.9% ) , followed by graft 12 ( 22.2% ) , internal 5 ( 9.3% ) and surgery 9 ( 16.7% ) .
thirty - six isolates ( 66.7% ) belonged to male patients and 18 ( 33.3% ) belonged to female patients .
the highest percentage ( 55.6% ) of resistance was observed against ceftazidime and imipenem with 30 of the isolates being resistant ; all isolates were sensitive to polymyxin b ( figure 1 ) .
twenty - eight ( 51.8% ) isolates tested in the study revealed resistance to all applied antibiotics except for polymyxin b. electrophoresis patterns of rapd - pcr for some isolates of studied p. aeruginosa are shown in figure 2 .
genetic relatedness of clinical isolates of the detected p. aeruginosa has been carried out using rapd - pcr assay , which showed 55% to 100% similarity ( figure 3 ) .
the number of bands in rapd typing varied from 2 to 12 , with the length of 150 bp to 4300 bp .
rapd fingerprinting results of p. aeruginosa based on  80% showed 39 different groups ( figure 3 ) . although the majority of isolates had unique fingerprint , group one composed of 3 isolates ( patients s48 , s29 , s31 ) .
isolate s29 was fully sensitive to applied antibiotics , whereas isolates s31 and s48 were resistance to applied antibiotics ( figure 3 ) .
groups 22 , 31 , 35 , and 36 , each consisted of two isolates , were also obtained from the same ward ( figure 3 ) .
based on 65% similarity level , eight main cluster , with most of the strains belonging to cluster 1 were detected ( figure 3 ) .
line 1 , 14 dna ladder ( 3000 - 100 bp ) , lines 2 - 13 showed 12 different rapd types .
abbreviations : amk , amikacin ; azt , aztreonam ; caz , ceftazidime ; cip , ciprofloxacin ; imp , imipenem ; mp , meropenem ; sp , sputum ; uc , urine ; wo , wound .
constant monitoring of antibiotic susceptibility profiles and genetic relatedness among bacterial infectious agents are essential steps for infection control ( 34 ) . to improve our understanding about p. aeruginosa antibiotic resistance and their distribution , 54 non - duplicate isolates of p. aeruginosa by using kirby - bauer s disk diffusion and rapd - pcr methods
p. aeruginosa involved in this study were highly resistant to different antibiotic families like , monobactams , cephalosporins , quinolones , and carbapenems ( figure 1 ) .
our isolates had the highest resistance rate to imipenem and meropenem ( 55.6% ) , which is higher than similar reports from iran ( 11 - 13 ) .
in addition , increased percentage of resistance to ciprofloxacin and amikacin ( 53.7% and 48.1% , respectively ) were observed in comparison with previous study ( 29% and 17% ) that have been performed in the same hospital ( 35 ) .
although , in our investigated hospital , carbapenems ( imipenem , meropenem ) along with amikacin are the most currently used antibiotics for the treatment of p. aeruginosa infections , results of this study revealed that prescription of them should be limited . in order to optimize treatment of p. aeruginosa infections in our hospital (
also it can be mentioned as a limitation of this study ) , determination of resistance to other antibiotics like piperacillin , piperacillin - clavulanic acid , ticarcillin , ticarcillin - clavulanic acid , gentamicin , and cefepime , are essential to be evaluated in future studies .
the finding of present study corresponds with the data presented by haeili and associates , which showed the lowest percentage of resistance ( 0% ) to polymyxin b ( 11 ) .
there are reports that pfge and mlst are more reliable methods for establishing clonal relatedness among p. aeruginosa strains , but because of the high cost , their utilization is limited , especially in developing countries ( 36 , 37 ) . allegedly , rapd typing is a valuable and useful method for the evaluation of genetic diversity among p. aeruginosa isolates ( 21 , 23 , 24 ) . out of various primers applied for rapd - pcr ,
primer 272 was reliable because of its higher discriminatory power and reproducible profiles ( 38 , 39 ) . in the present study , all 54 isolates were typeable by rapd - pcr ; moreover , rapd typing allowed us to reveal 54 unique finger prints among 54 clinical isolates of p. aeruginosa .
previous studies performed in iran showed different distribution of genotypes . for instance , in the study of salimi et al .
, by using rapd typing , only 8 different groups among 129 isolates of p. aeruginosa were reported ( 40 ) . in addition , in another study conducted by nanvazadeh et al .
9 groups among 50 clinical samples of p. aeruginosa were observed ( 41 ) . using limited source for recovering p. aeruginosa
although in a previous study the correlation between rapd type and p. aeruginosa infection was suggested , our finding is not in agreement with these data ( 42 ) .
furthermore , our data demonstrated a weak correlation between rapd and antibiotic resistance profiles ; it is supported by the results of other studies ( 34 , 43 ) . in a number of cases ,
the reason for this diversity may be attributed to the involved different antibiotic resistance mechanisms ( 5 - 7 ) .
moreover ; results of the independent studies showed that determination of antibiotic resistance pattern as phenotypic method for p. aeruginosa typing has low discriminatory power ( 34 , 43 ) . according to our data ,
the majority of isolates probably originated from host itself , but cross infection of p. aeruginosa is possible to occur in studied hospital .
for instance , isolates s48 , s29 , and s31 were recovered from urine of the patients in different wards or isolates 26 and 33 from the same ward and sample had genetic similarity ( based on rapd fingerprinting results ) . before taking any decision
, we think that clonal relatedness of obtained isolates should be confirmed by more discriminatory methods , such as pfge or mlst . in conclusion ,
prescription of common antibiotics ( imipenem and amikacin ) in our hospital due to high resistance rate must be restricted . according to our data , considerable genetic diversity exists among isolated p. aeruginosa
; it is possible that different sources of p. aeruginosa be involved in our hospital that can lead to host colonization . in order to accurately control the infection , other p. aeruginosa isolated from hospital environment
 background : pseudomonas aeruginosa is a well - known opportunistic pathogen , which affects hospitalized patients in different wards due to its natural resistance to drugs.objectives:the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran.materials and methods : out of 150 analyzed samples , 54 p. aeruginosa isolates were recovered and were subjected to antibiotic resistance patterns and genetic diversity determination by kirby - bauer s disk diffusion method and rapd - pcr , respectively.results:the highest percentage of resistance was observed against ceftazidime and imipenem with 30 ( 55.6% ) isolates ; meanwhile all isolates were sensitive to polymyxin b. twenty - eight ( 51.8% ) isolates revealed resistance to all applied antibiotics . 
 rapd - pcr ( random amplified polymorphic dna- polymerase chain reaction ) results showed 54 unique genotypes , which were divided into 39 clusters.conclusions:although different source of p. aeruginosa may involve in patient colonization , genetically related strains were isolated from different wards and or the same ward of the hospital . 
 our results pointed to the restriction of currently used antibiotics in studied hospital . 
 we hope that our results cast light on the control and transmission of the infection in the investigated hospital . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Pseudomonas aeruginosa Isolation and Identification
3.2. Antibiotic Susceptibility Test
3.3. Random Amplified Polymorphic DNA-Genotyping
4. Results
5. Discussion