clinical isolates of e. coli , klebsiella pneumoniae , and k. oxytoca were obtained from 19 us hospitals during phase iv ( 20022004 ) of project icare , which focused on nonoutbreak isolates of enterobacteriaceae with decreased susceptibility to extended - spectrum cephalosporins , fluoroquinolones , or carbapenems . the 19 hospitals were located in 13 states throughout the country and had an approximately equal mix of teaching and nonteaching medical centers . isolates were tested for susceptibility to the antimicrobial drugs listed in table 1 by using the broth microdilution reference method ( bmd ) described by the clinical and laboratory standards institute ( 7 ) . isolates were stored at 70c and were subcultured to trypticase soy agar plates containing 5% defibrinated sheep blood ( bd biosciences , sparks , md , usa ) > 2 times before testing . for each organism , bmd tests were inoculated by using a cell suspension equivalent to a 0.5 mcfarland standard . enterobacteriaceae isolate identifications provided by participating laboratories were confirmed by colony shape , spot tests ( 8) , and vitek gni+ cards ( biomrieux , durham , nc , usa ) . odds ratio ; ci , confidence interval . variables with p values  0.05 , in boldface , are considered significant . isolates were analyzed by polymerase chain reaction ( pcr ) amplification techniques to determine whether a class 1 integron was present . the primer sequences used were inti1-f : 5-tct cgg gta aca tca agg-3  and inti1-r : 5-agg aga tcc gaa gac ctc-3  . amplifications were performed in 10 l of taq pcr master mix ( qiagen , valencia , ca , usa ) , 1.5 mmol / l mgcl2 , 5 pmol / l each primer , and 1 g template dna ( 1 ) . dna was extracted with a genelite bacterial genomic dna kit ( sigma , saint louis , mo , usa ) . amplification specifications were as follows : 5 min at 94c followed by 35 cycles of 1 min at 94c , 1 min at 55c , and 30 s at 72c . . a statistical comparison of the frequencies of integron presence in e. coli and klebsiella spp . was conducted by using odds ratios and 95% confidence intervals . multivariate logistic regression analysis was conducted with 1 representative from each major class of drugs tested . data validation and analysis were performed by sas statistical software version 9.1 ( sas institute , cary , nc , usa ) . fit of the data to this model was evaluated by the hosmer - lemeshow  test . a positive test result for class 1 integrons was found for 181 ( 57% ) of the 320 bacterial isolates screened , including 103 ( 49% ) of 209 e. coli isolates and 78 ( 70% ) of 111 klebsiella spp . a positive test result in e. coli isolates was significantly associated with nonsusceptibity to gentamicin , tobramycin , ciprofloxacin , chloramphenicol , and trimethoprim - sulfamethoxazole ( table 1 ) . a positive test result in klebsiella isolates was significantly associated with nonsusceptibility to gentamicin , tobramycin , cefpodoxime , ceftazidime , and trimethoprim - sulfamethoxazole . multivariate analysis showed that the only drug variables associated with a positive test result for class 1 integrons in e. coli were nonsusceptibility to gentamicin and trimethoprim - sulfamethoxazole ( with a strong interaction between the 2 variables ) , while an inverse association was present between a positive integron test result and nonsusceptibility to cefepime ( table 2 ) . for klebsiella spp . , a positive association existed between a positive integron test result and nonsusceptibility to gentamicin and trimethoprim - sulfamethoxazole , as well as an inverse association between a positive integron test result and nonsusceptibility to aztreonam . * multivariate logistic regression with variables included for 1 drug of each major class that was tested . hosmer and lemeshow goodness - of - fit test statistic : e. coli total  = 10.852 , df = 8 , p = 0.21 ; klebsiella spp . total  = 4.538 , df = 8 , and p = 0.81 . or , odds ratio ; ci , confidence interval .  by  approximation .  significant interaction between these 2 variables ( p<0.001 ) . more than half of the selected isolates from us hospitals that we tested were positive for class 1 integrons . the prevalence of class 1 integrons in e. coli was 49% in selected non - outbreak isolates from hospitalized patients from 2002 to 2004 in our study , 52% in consecutive urinary tract isolates in 2001 in sweden ( 10 ) , and 15% in isolates from blood in hospitals in sweden in 1998 and 1999 ( 11 ) . the prevalence of integrons was 70% in klebsiella isolates from hospitalized patients in our study and 73% in extended - spectrum -lactamase ( esbl)producing klebsiella spp . these data can not be directly compared because of differences in selection criteria and testing procedures , but as a whole they suggest that prevalence of integrons in these bacteria in the united states is high , as it is in other areas of the world . carbapenem resistance was infrequent in the isolates we tested , so we could not address the association of integron carriage and metallo--lactamases that has been observed in other parts of the world ( 2 ) . our study found an inverse association between integron presence and nonsusceptibility to cefepime in e. coli and to aztreonam in klebsiella spp . , which indicates that resistance determinants for these drugs are not frequently carried by integrons . one possible reason for the lack of an association between nonsusceptibility to cefepime and integron carriage is the relatively recent approval of cefepime for use in the united states . ctx - m - type -lactamases are the most common resistance determinants in cefepime - resistant isolates worldwide , but these enzymes are just beginning to appear in the united states ( 13 ) . in phase iv of project icare , participating sites were asked to submit isolates that showed decreased susceptibility to fluoroquinolones , extended - spectrum cephalosporins , or carbapenems . we did not track whether isolates were from patients in intensive care units or not . thus , our isolates made up a convenience sample strongly associated with drug resistance mechanisms carried as integron gene cassettes and otherwise , and they are not necessarily representative of isolates from the united states in general . we did not examine carriage of > 1 integron , as documented in earlier studies ( 10 ) . in these selected isolates , the crude association between a positive test result for class 1 integrons and nonsusceptibility to a fluoroquinolone ( ciprofloxacin ) in e. coli , and nonsusceptibility to a -lactam ( ceftazidime ) in klebsiella spp this finding suggests that the mechanisms of resistance to fluoroquinolones and -lactams are associated with mechanisms of resistance to the drugs that remained independently associated with class 1 integrons ( aminoglycosides and trimethoprim - sulfamethoxazole ) , but for reasons unrelated to integron carriage . a previous study also found that ciprofloxacin resistance no longer predicted integron presence in enterobacteriaceae when multivariate analysis was conducted ( 1 ) . a spanish study ( 14 ) found no association between integron carriage and -lactam resistance in esbl - producing e. coli strains unless strains contained metallo--lactamases , which were infrequently encountered in our study population . a biologic mechanism for association of resistance to fluoroquinolones and aminoglycosides ( i.e. , the inactivation of ciprofloxacin by the aac ( 6)-ib - cr aminoglycoside acetylating gene ) has been recently reported ( 15 ) . in our study , both crude and multivariate associations between integron positivity and nonsusceptibility varied for the e. coli and klebsiella groups . this finding emphasizes that studies of this type must assess whether analysis should combine results for organisms of different genus and species into larger groups ( e.g. , enterobacteriaceae ) . when determinants encoding resistance to a variety of antimicrobial classes are contained within an integron , use of any of these agents may select for and enhance expression of the other determinants ( 2 ) . thus , in the example of our studied isolates , use of aminoglycosides may lead to spread of a multidrug - resistant bacterial strain such as k. pneumoniae . the differential association of class 1 integrons with resistance in the isolates we studied suggests that integrons facilitate the spread of antimicrobial drug resistance in the united states .
we examined escherichia coli and klebsiella spp . from us hospitals for class 1 integrons . 
 of 320 isolates , 181 ( 57% ) were positive ; association of integrons with resistance varied by drug and organism . 
 thus , determining integron epidemiology will improve understanding of how antibacterial resistance determinants spread in the united states .