Class 1 integrons in resistant Escherichia coli and Klebsiella spp., US hospitals.

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.

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Integrons are genetic elements, located on the bacterial chromosome or a plasmid, that often carry genetic determinants for antimicrobial drug resistance (1). The need for systematic epidemiologic studies of the role of integrons in antimicrobial drug resistance in bacteria has recently been emphasized (2). The prevalence of integrons is high among gram-negative isolates from patients in Europe (3,4), and some carry multiple integrons (3). Reports from Asian countries also have noted a high prevalence of class I integrons in gram-negative clinical isolates (5). Most of the resistance integrons found to date in clinical isolates of Enterobacteriaceae are class 1 integrons, which are highly associated with resistance to antimicrobial agents (2).

These data suggest that integrons are relatively common, especially among the Enterobacteriaceae, and that they contribute to the spread of antimicrobial drug resistance in healthcare settings. However, few studies from the United States have assessed the association between integron carriage and antimicrobial susceptibility patterns.

This study analyzes the association between class 1 integrons and resistance to selected antimicrobial agents in a convenience sample of Escherichia coli and Klebsiella isolates. Multivariate analysis was used to determine whether apparent associations were affected by interactions between variables. Isolates were submitted from hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) (6). The protocol has been approved as exempt by the institutional review board at Emory University.

The Study

Clinical isolates of E. coli, Klebsiella pneumoniae, and K. oxytoca were obtained from 19 US hospitals during phase IV (2002-2004) 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 non-teaching medical centers. Duplicate isolates from the same patient were excluded.

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 -70[degrees]C and were subcultured to trypticase soy agar plates containing 5% defibrinated sheep blood (BD BioSciences, Sparks, MD, USA) [greater than or equal to] 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 (bioMerieux, Durham, NC, USA). Differences were resolved by using reference biochemical tests (9).

Isolates were analyzed by polymerase chain reaction (PCR) amplification techniques to determine whether a class 1 integron was present. Integrons were detected by PCR amplification of a class 1 integrase-specific fragment of the IntI1 gene as previously described (1). 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 [micro]L of Taq PCR Master Mix (Qiagen, Valencia, CA, USA), 1.5 mmol/L Mg[Cl.sub.2], 5 pmol/L each primer, and 1 [micro]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 94[degrees]C followed by 35 cycles of 1 min at 94[degrees]C, 1 min at 55[degrees]C, and 30 s at 72[degrees]C. PCR products were analyzed by gel electrophoresis with 1.5% agarose gels. All PCRs included positive and negative controls.

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. Intermediate and resistant isolates were pooled as nonsusceptible for analysis. 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 [chi square] 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. isolates. 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.

Conclusions

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 [beta]-lactamase (ESBL)--producing Klebsiella spp. collected from 3 hospitals in Australia from 1989 to 1999 (12). These data cannot 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-[beta]-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 [beta]-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 [beta]-lactam (ceftazidime) in Klebsiella spp. failed to remain as independent variables in the multivariate model. This finding suggests that the mechanisms of resistance to fluoroquinolones and [beta]-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 [beta]-lactam resistance in ESBL-producing E. coli strains unless strains contained metallo-[beta]-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.

Acknowledgments

We thank the microbiology personnel at participating hospitals for sending the isolates included in this study.

Phase 5 of Project ICARE is supported in part by unrestricted grants to the Rollins School of Public Health of Emory University by Astra-Zeneca Pharmaceuticals, bioMerieux Incorporated, Elan Pharmaceuticals, and Pfizer Incorporated.

Ms Rao is a clinical pharmacy student at Midwestern University in Glendale, Arizona. Her research interests include the relationship of antimicrobial drug use and resistance in healthcare-associated bacteria.

References

(1.) Leverstein-van Hall MA, Blok HEM, Donders ART, Paauw A, Fluit AC, Verhoef J. Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis. 2003; 187:251-9.

(2.) Norrby SR. Integrons: adding another threat to the use of antibiotic therapy. Clin Infect Dis. 2005;41:10-1.

(3.) Sallen B, Rajoharison A, Desvarenne S, Mabilat C. Molecular epidemiology of integron-associated antibiotic resistance genes in clinical isolates of Enterobacteriaceae. Microb Drug Resist. 1995;1: 195-202.

(4.) Martinez-Freijo P, Fluit AC, Schmitz FJ, Grek VS, Verhoef J, Jones ME. Class I integrons in gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds. J Antimicrob Chemother. 1998;42:689-96.

(5.) Yu HS, Lee JC, Kang HY, Ro DW, Chung JY, Jeong YS, et al. Changes in gene cassettes of class 1 integrons among Escherichia coli isolates from urine specimens collected in Korea during the last two decades. J Clin Microbiol. 2003;41:5429-33.

(6.) McGowan JE Jr, Hill HA, Volkova NV, Lawton RM, Haber MJ, Tenover FC, et al. Does antimicrobial resistance cluster in individual hospitals? J Infect Dis. 2002;186:1362-5.

(7.) National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 6th ed. NCCLS document M7-A6. Wayne (PA): The Committee; 2003.

(8.) Forbes B, Sahm D, Weissfeld A. Enterobacteriaceae. Saint Louis (MO): Mosby; 1998.

(9.) Farmer JJ III. Enterobacteriaceae: introduction and identification. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manual of clinical microbiology. 8th ed. Washington: American Society for Microbiology; 2003. p. 636-53.

(10.) Grape M, Farra A, Kronvall G, Sundstrom L. Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant gram-negative bacteria. Clin Microbiol Infect. 2005; 11:185-92.

(11.) Heir E, Lindstedt BA, Leegaard TM, Gjernes E, Kapperud G. Prevalence and characterization of integrons in blood culture Enterobacteriaceae and gastrointestinal Escherichia coli in Norway and reporting of a novel class 1 integron-located lincosamide resistance gene. Ann Clin Microbiol Antimicrob. 2004;3:12.

(12.) Jones LA, McIver CJ, Kim MJ, Rawlinson WD, White PA. The aadB gene cassette is associated with blaSHV genes in Klebsiella species producing extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 2005;49:794-7.

(13.) Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48:1-14.

(14.) Machado E, Canton R, Baquero F, Galan JC, Rollan A, Peixe L, et al. Integron content of extended-spectrum-beta-lactamase-producing Escherichia coli strains over 12 years in a single hospital in Madrid, Spain. Antimicrob Agents Chemother. 2005;49:1823-9.

(15.) Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med. 2006;12:83-8.

Address for correspondence: John E. McGowan, Jr, Rollins School of Public Health, Emory University, 1518 Clifton Rd (Room 442), Atlanta, GA 30322, USA; email [e-mail]

Aarati N. Rao, * (1) Miriam Barlow, * (2) Leigh Ann Clark, ([dagger]) John R. Boring, III, * Fred C. Tenover, ([dagger]) and John E. McGowan, Jr *

* Emory University, Atlanta, Georgia, USA; and ([dagger]) Centers for Disease Control and Prevention, Atlanta, Georgia, USA

(1) Current affiliation: Midwestern University, Glendale, Arizona, USA

(2) Current affiliation: University of California at Merced, Merced, California, USA

Table 1. Crude association between nonsusceptibility to various
antimicrobial drugs and positive test result for class 1 integron in
Escherichia coli and Klebsiella spp. (Klebsiella pneumoniae and K.
oxytoca)

                                                      Klebsiella spp.
                               E. coli (n = 209),    (n = 111), OR (95%
Class/agent                       OR (95% CI) *            CI) *

Aminoglycosides
  Amikacin                      3.15 (0.32-30.79)    4.0 (0.86-18.51)
  Gentamicin                    6.04 (2.69-9.47)#    4.49 (1.80-11.22)#
  Tobramycin                    6.66 (3.01-10.23)#   6.0 (2.0-12.52)#
Fluoroquinolone
  Ciprofloxacin                 3.00 (1.40-6.42)#    1.33 (0.59-3.04)
3rd-generation cephalosporin
  Cefotaxime                    1.14 (0.63-2.07)     2.26 (0.99-5.19)
  Cefpodoxime                   1.02 (0.59-1.75)     2.61 (1.05-6.52)#
  Ceftazidime                   1.30 (0.73-2.31)     2.54 (1.10-5.88)#
4th-generation cephalosporin
  Cefepime                      0.35 (0.11-1.13)     1.87 (0.57-6.10)
Monobactam
  Aztreonam                     1.58 (0.87-2.89)     1.10 (0.48-2.53)
[beta]-Lactamase inhibitor
  Piperacillin-tazobactam       1.40 (0.75-2.62)     1.82 (0.80-4.14)
Others
  Chloramphenicol               2.33 (1.30-4.17)#    2.29 (0.97-5.42)
  Minocycline                   1.57 (0.90-2.76)     1.20 (0.53-2.71)
  Trimethoprim-
    sulfamethoxazole           12.24 16.28-23.86#    3.65 (1.57-8.62)#

Note: Values indicated with # are considered significant.

* OR, odds ratio; CI, confidence interval. Variables with p values
[less than or equal to] 0.05, in boldface, are considered significant.

Table 2. Association of nonsusceptibility to various antimicrobial
agents and presence of integrons in Escherichia coli and Klebsiella
spp. (Klebsiella pneumoniae and K. oxytoca) *

Antimicrobial drug                 Estimate       OR (95% CI)

E. coli
  Gentamicin ([double dagger])       1.16      3.19 (1.40-7.30)
  Ciprofloxacin                      0.31      1.37 (0.46-4.03)
  Ceftazidime                       -0.81      0.45 (0.14-1.45)
  Cefepime                          -1.77      0.17 (0.04-0.82)
  Piperacillin-tazobactam           -0.38      0.68 (0.28-1.66)
  Aztreonam                          1.19      3.29 (0.90-12.0)
  Chloramphenicol                    0.29      1.34 (0.60-2.99)
  Minocycline                       -0.11      0.89 (0.39-2.06)
  Trimethoprim-sulfamethoxazole
    ([double dagger])                2.56      12.9 (5.73-29.0)
  Source ([section])                 1.10      3.02 (1.23-7.45)
  Intercept                         -2.43
Klebsiella spp.
  Gentamicin                         1.44      4.23 (1.48-12.03)
  Ciprofloxacin                      0.34      1.40 (0.43-4.56)
  Ceftazidime                        0.92      2.51 (0.49-12.80)
  Cefepime                           0.05      1.05 (0.27-4.14)
  Piperacillin-tazobactam            0.02      1.02 (0.26-4.07)
  Aztreonam                         -1.64      0.19 (0.04-0.99)
  Chloramphenicol                   -0.57      0.57 (0.13-2.44)
  Minocycline                        0.13      1.14 (0.35-3.73)
  Trimethoprim-sulfamethoxazole      1.52      4.61 (1.39-15.29)
  Source ([section])                 0.56      1.75 (0.64-4.78)
  Intercept                         -0.28

Antimicrobial drug                 p value ([dagger])

E. coli
  Gentamicin ([double dagger])          0.006
  Ciprofloxacin                         0.573
  Ceftazidime                           0.180
  Cefepime                              0.027
  Piperacillin-tazobactam               0.407
  Aztreonam                             0.071
  Chloramphenicol                       0.476
  Minocycline                           0.791
  Trimethoprim-sulfamethoxazole
    ([double dagger])                  <0.001
  Source ([section])                    0.020
  Intercept                            <0.0001
Klebsiella spp.
  Gentamicin                            0.007
  Ciprofloxacin                         0.578
  Ceftazidime                           0.270
  Cefepime                              0.942
  Piperacillin-tazobactam               0.979
  Aztreonam                             0.048
  Chloramphenicol                       0.447
  Minocycline                           0.835
  Trimethoprim-sulfamethoxazole         0.012
  Source ([section])                    0.275
  Intercept                             0.591

* 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 [chi square] = 10.852,
cif= 8, p = 0.21; Klebsiella spp. total [chi square] = 4.538, df = 8,
and p = 0.81. OR, odds ratio; CI, confidence interval.

([dagger]) By [chi square] approximation.

([double dagger]) Significant interaction between these 2 variables
(p<0.001).

([section]) Nonurine source vs. urine source (reference).