The presence of aac(6′)-Ib-cr is associated with decreased susceptibility to aminoglycosides (kanamycin, amikacin, and tobramycin) and to norfloxacin and ciprofloxacin (9). This allelic variant of aac(6′)-Ib was found to be linked to the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-15 in isolates from many countries (4, 6, 7), while association of aac(6′)-Ib with the blaCTX-M-2 ESBL gene has been widely reported in Uruguay and Argentina (3, 11).
In this work we looked for the presence of aac(6′)-Ib and the aac(6′)-Ib-cr variant and their putative ESBL coresistance markers in fecal isolates of enterobacteria resistant to ciprofloxacin and/or ceftazidime from inpatients in an intensive care unit (ICU) in Montevideo, Uruguay.
From 1 March to 31 October 2006, 106 patients were admitted to this ICU and followed daily until discharge. Rectal swabs obtained at 1, 4, 7, 10, 13, and 16 days after admission were plated on MacConkey agar plus ceftazidime (4 mg/liter) or ciprofloxacin (2 mg/liter). Enterobacterial isolates were identified by classical methods, including only the first isolate of each bacterial species per patient in this study.
Antibiotic resistance profiling, screening, and confirmatory testing for ESBL detection were performed by disk diffusion assay, and results were interpreted following the CLSI guidelines (2).
A total of 58/106 patients (55.2%) were colonized with ciprofloxacin- and/or ceftazidime-resistant enterobacteria, and 68 isolates were included in this study. Of these, 48 were resistant to gentamicin and 24 to amikacin (Table 1).
TABLE 1.
Main characteristics of the 68 studied isolatesa
| Species | No. of isolates
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total | Positive for ESBLb | Resistant to drug:
|
Positive for aac(6′)-Ib | With major resistance phenotyped,e:
|
||||||||
| CAZ | FOX | GEN | AMK | CIP | SXT | CAZ-GEN | CIP-GEN | CAZ-CIP-GEN | ||||
| E. coli | 22 | 6 | 6 | 0 | 12 | 2 | 21 | 17 | 4/2c | 0 | 7 (1) | 5/2c (3) |
| Klebsiella pneumoniae | 13 | 7 | 11 | 10 | 8 | 5 | 7 | 7 | 8 | 2 (2) | 0 | 6 (6) |
| Enterobacter spp. | 27 | 6 | 27 | 27 | 23 | 17 | 19 | 22 | 17 | 5 (2) | 0 | 18 (15) |
| Other | 6 | 3 | 5 | 5 | 5 | 0 | 2 | 5 | 1 | 3 (1) | 1 | 1 |
| Total | 68 | 22 | 49 | 42 | 48 | 24 | 49 | 51 | 30/2c | 10 (5) | 8 (1) | 30 (24) |
Abbreviations: CAZ, ceftazidime; FOX, cefoxitin; GEN, gentamicin; AMK, amikacin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole.
The screening test for ESBL detection was performed according to CLSI recommendations.
The number after the shill is the number of positive cr variants.
Numbers in parentheses are numbers of isolates positive by PCR for the presence of aac(6′)-Ib.
Phenotypes: CAZ-GEN, resistance to ceftazidime and gentamicin and susceptibility to ciprofloxacin; CIP-GEN, resistance to ciprofloxacin and gentamicin and susceptibility to ceftazidime; CAZ-CIP-GEN, resistance to ceftazidime, gentamicin, and ciprofloxacin.
All aminoglycoside-resistant isolates were screened for aac(6′)-Ib by PCR; amplicons were analyzed by restriction with BstF5I, as described by Park et al. (8). PCR products that were not digested by the enzyme [tentatively assigned to aac(6′)-Ib-cr] were confirmed to contain aac(6′)-Ib-cr by double-strand sequencing. Only two Escherichia coli isolates were positive for aac(6′)-Ib-cr detection.
Recalling the observed links between blaCTX-M-15 and aac(6′)Ib-cr (4, 6, 7) and between aac(6′)Ib and blaCTX-M-2, the two aac(6′)-Ib-cr-positive isolates were further analyzed by PCR to detect CTX-M-1 and CTX-M-2 group ESBL genes using previously described primers (3, 5). Both isolates were positive only for CTX-M-1 group genes, identified as blaCTX-M-15 after sequencing.
Both isolates were obtained at the time of patient admission into the ICU and showed identical pulsed-field gel electrophoresis patterns (10). Both patients were previously hospitalized before ICU admission, suggesting that this strain could be endemic in the hospital, where it could be horizontally transferred. All the other E. coli isolates yielded different pulsotypes (data not shown) compared with these.
PCR assays for the detection of class 1 integrons and ISCR1 elements were performed according to the method of Di Conza et al. (3). Both isolates carried a class 1 integron containing the dfr17 and aadA5 gene cassettes, while ISCR1 elements were not detected.
So far we have not been able to transfer these resistance genes, either by transformation or by conjugation.
This is the first report of aac(6′)-Ib-cr in Uruguay. In accordance with a previous report (6), blaCTX-M-15 and aac(6′)-Ib-cr do not seem to be associated with class 1 integrons. Demonstration of a link to IS26 as previously reported (1) is pending.
Acknowledgments
This work was partially supported by grants from S/C/OP/76/30 PDT (Programa de Desarrollo Tecnológico, Ministerio de Educación y Cultura Uruguay) to R.V. and also by grants from CSIC (Comisión Sectorial de Investigación Científica Uruguay) to N.F.C. Part of this work was also supported by grants LSHM-CT-2003-503335 from the European Community and BFU2006-04574 from Ministerio de Educación y Ciencia, España, to J.A.A. and grants from UBACYT and ANPCYT and a Carrillo-Oñativia fellowship to G.G.
Footnotes
Published ahead of print on 12 November 2007.
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