acinetobacter baumannii is an aerobic , nonfermentative gram negative coccobacillus that can survive for prolonged periods in the environment and can cause nosocomial infections , ( urinary tract and wound infections ) .
extensive use of antimicrobial agents in clinical cases has contributed to the appearance of resistant a. baumannii strains .
increasing resistance to carbapenems in a. baumannii infections in the past decade is creating therapeutic challenges .
one of the characteristics of pathogenic bacteria is that they contain virulence factors ( vfs ) .
nonadhesive vfs include : diverse siderophores , such as ; yersiniabactin ( fyua ) , aerobactin ( iuta ) , invasins ( ibea ) , serum resistance ( trat ) , and colicin v production ( cvac ) .
the purposes of this work was to detect the genes codifying nonadhesive vfs , and to evaluate the frequency distribution of nonadhesive vfs in carbapenemase - producing a. baumannii isolated from clinical samples .
one - hundred four samples were isolated from patients hospitalized in three hospitals affiliated with kermanshah university of medical sciences , iran during march 2011-march 2013 .
the strains were identified as a. baumannii by conventional biochemical tests and api 20ne kit ( version 6.0 , bio - mrieux , france ) .
susceptibility to carbapenem was performed according to the standard clinical and laboratory standards institute guidelines to check their resistance to carbapenems using imipenem ( 10 g ) and meropenem ( 10 g ) ( mast , merseyside , uk ) .
escherichia coli mk1 and mk2 were used for ibea and fyua as a positive control . for vf trat , cvac and iuta
one amplified a polymerase chain reaction ( pcr ) product of each gene sent to sinaclon company for sequencing and the results were used as the positive control after nblast alignment .
bacteria were harvested from 1 ml of an overnight broth culture , suspended in 200 l of sterile water , and incubated at 100c for 10 min .
following centrifugation of the lysate , a 150 l sample of the supernatant was stored at 20c as a template dna stock .
each reaction mixture ( 15 l ) contained 2 l of dna , 0.5 mm of each primer , 1 u of taq dna polymerase ( sinaclon ) , the four deoxynucleoside triphosphates ( each at 200 m ) and 1.5 mm mgcl2 .
pcr was performed under predenaturation at 94c for 5 min , followed by 35 cycles of 94c for 45 s , at each specific annealing temperature [ table 1 ] for 30 s , and 72c for 45 s ending with a final extension step at 72c for 5 min .
the pcr product was run and visualized on 1.5% agarose gels then were stained with ethidium bromide .
primers to vfs used in the polymerase chain reaction a statistical comparison of the frequencies of nonadhesive vfs in carbapenemase - producing a. baumannii isolates was conducted by chi - squared test ( variables were analyzed by chi - square test ) .
one - hundred four samples were isolated from patients hospitalized in three hospitals affiliated with kermanshah university of medical sciences , iran during march 2011-march 2013 .
the strains were identified as a. baumannii by conventional biochemical tests and api 20ne kit ( version 6.0 , bio - mrieux , france ) . susceptibility to carbapenem
was performed according to the standard clinical and laboratory standards institute guidelines to check their resistance to carbapenems using imipenem ( 10 g ) and meropenem ( 10 g ) ( mast , merseyside , uk ) .
escherichia coli mk1 and mk2 were used for ibea and fyua as a positive control . for vf trat , cvac and iuta
one amplified a polymerase chain reaction ( pcr ) product of each gene sent to sinaclon company for sequencing and the results were used as the positive control after nblast alignment .
bacteria were harvested from 1 ml of an overnight broth culture , suspended in 200 l of sterile water , and incubated at 100c for 10 min .
following centrifugation of the lysate , a 150 l sample of the supernatant was stored at 20c as a template dna stock .
each reaction mixture ( 15 l ) contained 2 l of dna , 0.5 mm of each primer , 1 u of taq dna polymerase ( sinaclon ) , the four deoxynucleoside triphosphates ( each at 200 m ) and 1.5 mm mgcl2 .
pcr was performed under predenaturation at 94c for 5 min , followed by 35 cycles of 94c for 45 s , at each specific annealing temperature [ table 1 ] for 30 s , and 72c for 45 s ending with a final extension step at 72c for 5 min .
the pcr product was run and visualized on 1.5% agarose gels then were stained with ethidium bromide .
a statistical comparison of the frequencies of nonadhesive vfs in carbapenemase - producing a. baumannii isolates was conducted by chi - squared test ( variables were analyzed by chi - square test ) .
one - hundred four a. baumannii specimens were collected from three hospitals in kermanshah ( iran ) .
fifty isolates were resistant to imipenem and meropenem and also positive for one of the carbapenemase - encoding genes ( blaoxa-23-like , blaoxa-24-like ) ; these isolates were detected as carbapenemase - producing isolates for future analysis .
the carbapenemase resistant bacteria were isolated from 35 ( 70% ) males to 15 ( 30% ) females , and the distributions of carbapenemase - producing a. baumannii , according to age , are shown in figure 1 .
frequencies of nonadhesive vfs include : serum resistance ( trat ) , colicin v ( cvac ) , and aerobactin ( iuta ) , were ; 40 isolates ( 80% ) , 17 isolates ( 34% ) , and 8 isolates ( 16% ) , respectively .
however , the genes fyua , ibea , and pathogenicity - associated islands ( pai ) , codifying for ; yersiniabactin , invasion and pais , respectively , were not detected by means of a pcr with specific primers .
eight ( 16% ) isolates carried both trat and cvac ( p = 0.27 ) , 6 ( 12% ) isolates carried trat and iuta ( p = 0.18 ) , and 4 ( 8% ) isolates carried cvac and iuta ( p = 0.41 ) .
distribution of trat , cvac and iuta did not correlate with gender [ table 2 ] .
distribution of carbapenemase - producing acinetobacter baumannii according to the age of patients patterns of agarose gel electrophoresis showing polymerase chain reaction amplification products for the nonadhesive virulence factors genes .
( a ) lane 1 : trat ( 290 bp ) ; lane 3 : cvac ( 680 bp ) ; lane 5 : iuta ( 300 bp ) ; lane 2 , 4 , 6 : negative control .
( b ) lane 2 : fyua ( 785 bp ) ; lane 4 : ibea ( 170 bp ) ; lane 3 and 5 : negative control distribution of trat , cvac and iuta according to sex
treatment of infections with opportunistic pathogens such as a. baumannii has been a great challenge .
carbapenems are the first - line of treatment of acinetobacter infections , but nowadays resistance to them is reported . in this study , 50 isolates ( 48% ) were recognized as carbapenemase - producing phenotypes out of 104 clinical isolates of a. baumannii .
park et al . reported resistance to imipenem and meropenem , 31.7% and 34.9% respectively from 63 isolates of acinetobacter in south korea .
resistant rate to carbapenem in a. baumannii isolates investigated in this study is different compared to other studies , which may be due to geographical differences in isolates .
fifty carbapenemase - producing isolates were examined in terms of having six nonadhesive vfs including trat ( serum resistance ) , cvac ( colicin v ) , iuta ( aerobactin ) , ibea , ( invasion ) , fyua ( yersiniabactin ) and pai .
frequency of trat , cvac and iuta factors were 40 ( 80% ) , 17 ( 34% ) and 8 ( 16% ) isolates , respectively .
vfs cause infection in most gram negative pathogenic bacteria . of these , nonadhesive vfs play a key role in bacterial survival in special conditions such as in human blood and exposure to serum and poor iron environments that cause diseases . compared to other gram - negative pathogens ,
serum resistance ( trat ) is one of the nonadhesive factors investigated in this study .
studies indicate that serum resistance is the ability of the bacterial cell to resist the lytic effects of serum and to invade and survive in the human blood stream .
2012 ) studied 14 samples of acinetobacter and indicated that 7 isolates ( 50% ) had serum resistance phenotypically .
king et al . in the usa found that from 7 a. baumannii isolates , 3 isolates ( 43% ) had serum resistance phenotypically .
the results obtained from carbapenemase - producing a. baumannii isolates in this study show that genotypic frequency of trat gene is higher than in other studies .
of course , this difference could be due to phenotypic and genotypic detection techniques for these vfs .
there is also a direct relationship between serum resistance and biofilm formation , and hence they increase bacterial pathogenicity . as a result ,
considering the existence of 80% trat gene in this study , more pathogenicity is expected for these isolates .
another vf described in a. baumannii is the production of siderophores by isolates growing under iron limiting conditions .
these bacteria are capable to express high - affinity iron uptake systems composed by siderophores . in braun study , all a. baumannii strains were able to grow under iron limiting conditions , and they concluded that this bacterium produces siderophores . unlike our study , the genes iuta and fyua from iron uptake system of e. coli and yersinia sp .
. considering the frequency of iuta gene in this study , it seems that iuta siderophore unlike fyua in acinetobacters isolated from sputum / tracheal secretion , is more important vf compared to urinary isolates .
another gene that was positive in this study is cvac , which was observed in 34% of cases .
cvac , which encodes colicin v , have been proposed to confer enhanced virulence through their carriage of other specific vfs , including the aerobactin system and serum survival genes , such as trat .
carbapenemase - producing a. baumannii strains are among the most important agents of nosocomial infections in our hospital and worldwide .
three vfs of trat , cvac and iuta with frequency of 80% , 34% , and 16% , respectively , revealing a role in the pathogenesis of these isolates and also may be have the most impact on existence for long time by carbapenemase - producing a. baumannii in environment of hospital .
frequent screening for these vfs may contribute to better infection control measures and to the investigate new treatment options .
 background : acinetobacter baumannii which is a gram - negative bacterium can cause several different infections . 
 the appearance of carbapenemase - producing a. baumannii in recent years has made the treatment process more difficult . 
 the identification of virulence factors ( vfs ) , such as nonadhesives in a. baumannii , helps to fight against related infections.materials and methods : a total of 104 samples from teaching hospitals in kermanshah , iran , were collected during a 24 months period ( 2011 - 2013 ) . 
 sample identification was first carried out by biochemical tests , and then their susceptibility to carbapenems was determined using the kirby  bauer method . for confirmation of carbapenemase - producing a. baumannii , polymerase chain reaction ( pcr ) 
 was done for carbapenemase - encoding genes . 
 in addition , the frequency of nonadhesive vfs in carbapenemase - producing isolates was determined by pcr.results:there were 50 isolates that were identified as carbapenemase - producing a. baumannii . 
 the pcr results showed ; 40 isolates ( 80% ) for trat , 17 isolates ( 34% ) for cvac , and 8 isolates ( 16% ) for iuta , and these encode serum resistance , colicin v and aerobactin , respectively . 
 no significant correlation was observed between these three genes.conclusions:the mechanism of a. baumannii virulence has always been in question . 
 the role of vfs has also been recognized in other gram - negative bacteria . according to the prevalence of trat , cvac and iuta , as nonadhesive vfs 
 , we can suggest that they could be the main mechanism of carbapenemase - producing a. baumannii pathogenesis . 
INTRODUCTION
MATERIALS AND METHODS
Bacterial collection
Identification and antimicrobial susceptibility testing
Polymerase chain reaction
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest