Spread of Escherichia coli Strains with High-Level Cefotaxime and Ceftazidime Resistance between the Community, Long-Term Care Facilities, a
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《微生物临床杂志》
Antibiotic Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
Microbiology Department, Hospital Gregorio Maraon, Madrid, Spain
Microbiology Department, Fundacion Hospital de Alcorcon, Alcorcon, Madrid, Spain
Microbiology Department, Hospital Severo Ochoa, Leganes, Madrid, Spain
Microbiology Department, Centro de Especialidades Argüelles, Hospital Puerta de Hierro, Madrid, Spain
Consejo Superior de Investigaciones Científicas, Madrid, Spain
ABSTRACT
A total of 151 Escherichia coli strains resistant to cefotaxime and ceftazidime were isolated during a prospective surveillance study. These strains were characterized by clinical, microbiological, and molecular analyses and were distributed into four clusters of 103, 11, 6, and 5 isolates, along with 25 unrelated strains. The principal cluster was isolated from urine, wound, blood, and other samples in three hospitals, eight nursing homes, and a community healthcare center. This cluster was associated with both nosocomial (65%) and community-acquired (35%) infections. Most strains were resistant to ciprofloxacin, gentamicin, tobramycin, cefepime, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole but were susceptible to imipenem. All isolates from the four clusters expressed the extended-spectrum -lactamase (ESBL) CTX-M-15. This enzyme was also present in 8 (30.8%) of the 26 unrelated isolates. The other ESBLs, CTX-M-14 and CTX-M-32, were detected in five and seven cases, respectively, but they were detected in individual E. coli isolates only. In three clusters, blaCTX-M-15 alleles were linked to an ISEcp1-like element, while in eight strains of cluster II an IS26 element preceded the blaCTX-M-15 allele. An additional pool of resistance genes included tetA, drfA14 or dfrA17, sul1 or sul2, aac(6')Ib, and aac(3)IIb. All except one of the 27 isolates tested for genetic virulence markers harbored the same three virulence genes: iutA and fyuA (siderophores), and traT (serum survival factor). Epidemic or occasional isolates of cefotaxime- and ceftazidime-resistant E. coli can spread between distinct health facilities including hospitals, community health centers, and long-term care centers.
INTRODUCTION
The production of extended spectrum -lactamases (ESBLs) is one of the major sources of resistance to extended-spectrum cephalosporins in Enterobacteriaceae (16). Classically, plasmid-mediated ESBL enzymes have been of the TEM and SHV types, but in recent years CTX-M-type ESBLs have been increasingly found throughout the world (1). CTX-M enzymes predominantly hydrolyze cefotaxime but are weakly active against ceftazidime. However, some ESBLs of the CTX-M family display increased hydrolytic activities against ceftazidime, as is the case for CTX-M-15 (24) and CTX-M-32 (4).
In recent years, ESBL production in Enterobacteriaceae, particularly Escherichia coli, has significantly increased in several countries, including Spain (6, 21, 30). This increase is primarily due to the spread of CTX-M-type ESBLs (25).
ESBL dissemination in E. coli is usually due to plasmid transmission between unrelated strains (9), while clonal spreading is more frequent in other Enterobacteriaceae, such as Klebsiella pneumoniae (23). However, outbreaks of ESBL-producing E. coli clones have recently been described (2, 15, 18, 19, 32). In Spain, the proportion of E. coli CTX-M producers is rapidly increasing in both nosocomial and community-acquired infections (21, 25).
We have observed an apparent and alarming increase of cefotaxime- and ceftazidime-resistant strains among the E. coli isolates submitted to our laboratory. These events prompted the present study in which we describe the emergence and spread of high-level cefotaxime- and ceftazidime-resistant E. coli isolates, their complex molecular epidemiology, and the characterization of several clusters of multidrug-resistant CTX-M-15-producing E. coli isolates. Epidemic strains were detected in patients admitted to hospitals and nursing homes for the elderly, as well as in patients attending an outpatient community healthcare center.
MATERIALS AND METHODS
Study design. A prospective surveillance study of infections caused by ESBL-producing E. coli is currently ongoing in the Autonomous Community of Madrid (Spain). Three hospitals located in three separate geographic areas participated in the study and included Hospital Gregorio Maraon (estimated catchment population of 650,000), Hospital Fundacion de Alcorcon (estimated catchment population of 250,000, including eight associated nursing homes), Hospital Severo Ochoa de Leganes (estimated catchment population of 380,000), and a community healthcare center, Centro de Especialidades Argüelles. All collected the initial E. coli isolates that displayed ESBL production and sent them to the Centro Nacional de Microbiología, a public health reference institution, to confirm ESBL production and to study the molecular epidemiology of the isolates and the ESBL type. Here we describe the analysis of all ESBL-producing E. coli strains, isolated between January 2004 and August 2005, which exhibited resistance to cefotaxime (>32 μg/ml) and ceftazidime (16 μg/ml).
Infections and patient characteristics. The participating institutions collected the following information: personal patient data (code, age, sex, clinical diagnosis, and risk factors), hospital and departmental data, and isolate data (clinical sample and antimicrobial susceptibility). This information was entered into the Whonet software program, a free microbiological database (WHO Collaborating Center for the Surveillance of Antibiotic Resistance).
Patients with acquired community infections were those presenting at community health care centers and those who had a positive culture at the time of or within 48 h of hospitalization; in both cases, patients had no previous contacts with hospitals or long-term care facilities in the last 2 weeks.
Antimicrobial susceptibility testing and ESBL production detection. E. coli isolates were identified and antibiotic susceptibility was tested according to standard microbiological procedures performed in each clinical laboratory. At the Centro Nacional de Microbiología, isolates were subcultured in both Columbia blood agar and MacConkey agar to ensure viability and purity. The identification of all isolates was confirmed according to standard microbiological methods; susceptibility testing was initially carried out by microdilution (BD Phoenix Automated Microbiology System; Becton Dickinson Diagnosis, Sparks, MD) and the disk diffusion reference method (5). The antimicrobial agents tested were as follows: ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefoxitin, cefotaxime, ceftazidime, cefepime, amikacin, gentamicin, tobramicin, ciprofloxacin, imipenem, and trimethoprim-sulfamethoxazole. ESBL production was confirmed as described by the Clinical Laboratory Standards Institute (5); inhibition zones obtained by using disks that contained cefotaxime (30 μg) and ceftazidime (30 μg) were compared to those containing cefotaxime-clavulanic acid (30 and 10 μg) and ceftazidime-clavulanic acid (30 and 10 μg) (Oxoid, Madrid, Spain), respectively. In addition, the cefotaxime and cefotaxime-clavulanic acid MICs, as well as for ceftazidime and ceftazidime-clavulanic acid, were determined by the E-test method (AB-Biodisk, Solna, Sweden). Antimicrobial susceptibility results were interpreted according to the breakpoints recommended by the Clinical Laboratory Standards Institute (5). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains.
Molecular epidemiology. The molecular epidemiology of E. coli isolates that were resistant to cefotaxime and ceftazidime was determined by pulsed-field gel electrophoresis (PFGE). Total bacterial DNA was digested with XbaI (MBI Fermentas, Vilnius, Lithuania), and DNA fragments were separated on a 1% agarose gel in 0.5x Tris-borate-EDTA buffer by using the CHEF Mapper apparatus (Bio-Rad, Madrid, Spain). The conditions were as follows: 14°C, 6 V/cm, pulse of 2 to 54 s, and 24 h. Gels were stained with ethidium bromide and photographed under UV light. Genetic similarity was calculated by the unweighted pair group method using arithmetic averages and presented in a dendrogram. Similarity was calculated by using Dice coefficients with a tolerance of 1.8% (Fingerprinting II Software; Bio-Rad). Well-resolved bands that corresponded to fragments exceeding 48.5 kb were included in the computer analysis.
Isoelectric focusing. Isoelectric focusing was carried out on a total of 73 E. coli isolates representing the major clones identified by PFGE. Bacteria, exponentially growing at 37°C in Luria-Bertani medium, were harvested, and cell-free lysates were prepared by sonication. Isoelectric focusing was performed by applying this crude extract to Phast gels (pH = 3 to 9) in a Phastsystem apparatus (Pharmacia AB, Uppsala, Sweden) (10). -Lactamases with known pI values of 5.9, 5.4, 7.6, and 8.1 were used in parallel as controls. Gels were stained with 500 μg of nitrocefin (Oxoid)/ml to identify -lactamase bands.
Molecular analysis of ESBLs. Based on both antibiotic resistance profiles and pI values, molecular identification of -lactamases was carried out by PCR amplification and DNA sequencing. All -lactamases detected by isoelectric focusing were characterized by molecular methods. Genomic DNA from wild-type isolates was used as a template for PCR. ESBL amplification was performed with the appropriate primers and cycling conditions for the TEM, SHV, CTX-M, OXA-1, OXA-2, and OXA-10 -lactamase groups, as described elsewhere (3, 22, 28). The following primers were designed to amplify and sequence the blaCTX-M-15 gene under standard conditions (CTX-M-15-F, 5'-GTT AAA AAA TCA CTG CG-3'; CTX-M-15-R, 5'-CAA ACC GTT GGT GAC G-3'). PCR products were separated on 0.8% agarose gels, stained with ethidium bromide, visualized under UV light, further purified by using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany), and sequenced by using an ABI Prism 377 automated sequencer (Perkin-Elmer, Norwalk, CT).
Insertion sequences. Linkage of blaCTX-M-15 alleles with insertion sequences (IS) was investigated; ISEcp1-like elements, previously implicated in their expression and mobilization (13), were confirmed with the primers PROM+ and PRECTX-M-3B as described previously (24, 32). In addition, specific primers were used to amplify a 400-bp fragment spanning the link between IS26 (inserted between ISEcp1 and blaCTX-M-15) and blaCTX-M-15; this fragment is believed to be characteristic of the epidemic E. coli strain A, a CTX-M-15 producer from the United Kingdom (32).
Identification of additional resistance genes. Based on antibiotic resistance profiles, additional mechanisms of antibiotic resistance were studied in 10 multiresistant CTX-M-15-producing isolates. Molecular identification of sul1, sul2, tetA, aac(6')Ib, aac(3)IIb, dfrA14, and dfrA17 genes was carried out by PCR amplification and DNA sequencing with primers previously described (8, 14, 15, 20). In addition, the acrR gene of the AcrAB efflux system was amplified, including the promoter-operator region, as described elsewhere (31).
Virulence factors. A total of 27 isolates from different clinical samples belonging to cluster I (12 from urine, 9 from blood, 5 from wounds, and 1 from the respiratory tract) and from different geographical sources (15 from three hospitals, 8 from the community, and 4 from four long-term care facilities) were evaluated for the presence of 10 genes encoding putative virulence factors characteristic of extraintestinal pathogenic E. coli using PCR as described previously (11). These genes included those encoding S fimbriae (sfaS), F1C fimbriae (focG), M blood group antigen-specific M fimbriae (bma), glucosaminyl-specific G fimbriae (gaf), Dr family adhesins (afa/dra), toxin associated with extraintestinal pathogenic E. coli (cnf1), siderophores as aerobactin (iutA), yersiniabactin (fyuA), the serum survival gene (traT), and the invasion of brain endothelium gene (ibeA).
Statistical analyses. Differences in the prevalence of antibiotic resistance between different groups were assessed by using the Fisher exact test. Association was determined by calculation of the odds ratio (OR) with 95% confidence intervals (CI). The null hypothesis was rejected for P values of <0.05. Statistical analyses were performed by using GraphPad Prism version 3.02 software (GraphPad Software, Inc., San Diego, CA).
RESULTS
Clinical isolates and population study. From January 2004 to August 2005, a total of 525 unduplicated E. coli ESBL producers were collected. Of these isolates, 151 (28.8%) were simultaneously resistant to cefotaxime and ceftazidime. A total of 65 (43%) were from males, 84 (55.6%) were from females, and 2 were from patients whose the gender was not reported. A total of 15 (9.9%) were from children 14 years of age, 21 (13.9%) were from 15- to 64-year-old patients, and 101 (66.9%) were from patients of 65 years; in 14 cases, the patient's age was unknown.
Of the 151 E. coli isolates, 76 (50.3%) were isolated from the urinary tract, 30 (19.9%) were from wounds, and 22 (14.6%) were from blood; the remaining 23 cases were isolated from other clinical samples (15.2%). Ninety-eight of the patients (64.9%) were admitted into different hospital departments: 33 (21.8%) to internal medicine, 16 (10.6%) to the emergency room, 12 (7.9%) to pediatric units, 7 (4.6%) to surgery, 5 (3.3%) to the intensive care unit, and 25 (16.5%) to other departments. Twenty-six patients (17.2%) came from long-term care facilities for elderly people, and twenty-one patients (13.9%) came from the community. In six cases, this information was not available.
In total, 92 isolates (60.9%) produced nosocomial infections (including those from nursing homes), 55 (36.4%) produced community-acquired infections, and for 4 isolates this information is unknown.
The clinical diagnoses included urinary tract infections (76 cases or 50.3%), wound infection (30 cases [19.9%]), sepsis (22 cases [14.6%]), pneumonia (7 cases [4.6%]), peritonitis (3 cases [2%]), and abscess (2 cases [1.3%]).
Seventy-nine patients (52.3%) had predisposing underlying conditions, with twenty-one of these being respiratory and/or cardiac disease (13.9%). Also among these conditions were 16 cases of impaired immunity (10.6%) (diabetes, 6; tumoral pathology, 4; transplantation, 3; human immunodeficiency virus infection, 2; rheumatic arthritis, 1), 11 cases of cognitive disorders (7.3%), 7 cases of urinary diseases (4.6%) (including 3 with a vesical catheter), 6 cases of premature birth (4%), and 4 cases of liver pathology (2.6%).
FIG. 1. Monthly distribution of total cases of E. coli isolates belonging to cluster I and other isolates resistant to cefotaxime and ceftazidime in two participating hospitals during 2004.
Antimicrobial susceptibility. Data on antimicrobial susceptibility for all 151 E. coli isolates resistant to cefotaxime and ceftazidime are provided in Table 1. The prevalences of antimicrobial resistance, calculated according to the MICs, were as follows: 59.9% for amoxicillin-clavulanic acid, 38.8% for piperacillin-tazobactam, 98% for cefepime, 8% for cefoxitin, 89.3% for ciprofloxacin, 82% for cotrimoxazole, 70% for gentamicin, 82.7% for tobramycin, and 1.3% for amikacin (Table 1).
Compared to nosocomial infections, community-acquired infections exhibited a lower resistance to gentamicin (56.4% versus 71%; P = 0.001; OR = 0.38, 95% CI = 0.18 to 0.79) and tobramycin (72.2% versus 87.6%; P = 0.025; OR = 0.36, 95% CI = 0.15 to 0.86). Most of the isolates were resistant to the majority of antibiotics tested. As shown in Table 2, 56 of the isolates (37.1%) exhibited multiresistance pattern A, being susceptible to amikacin, cefoxitin, and imipenem. Eleven of the isolates (7.3%) were classified as having multiresistance pattern B, being susceptible to amikacin and imipenem. Eleven of the isolates (7.3%) were susceptible to amikacin, cefoxitin, imipenem, and gentamicin (pattern C).
Molecular epidemiology. Cluster analyses of DNA fingerprinting performed on the 151 isolates is shown in Fig. 2. XbaI did not digest the DNA of one isolate.
FIG. 2. Dendrogram that illustrates the genetic relationship of 150 ESBL-producing E. coli isolates resistant to cefotaxime and ceftazidime. , isolate identification number.
Using PFGE analysis, four clusters of isolates were detected that exhibited a genetic relatedness of 85 to 100% (Fig. 2). A total of 103 isolates made up cluster I, 11 isolates made up cluster II, 6 isolates made up cluster III, and 5 isolates made up cluster IV. The remaining 25 isolates had a genetic similarity of <85% (Fig. 2) and were considered unrelated. Cluster I was distributed among all participant centers and included nosocomial (65%) and community (35%) isolates.
Clusters II and IV were distributed among three of the participant centers, whereas the six isolates of cluster III belonged exclusively to one of the centers.
Of the cluster I isolates, 3 (2.9%) were from children of 14 years of age, 13 (12.6%) were from 15- to 64-year-old patients, and 78 (75.7%) were from patients of 65 years; in nine cases, the patient's age was unknown (Fig. 3A). These isolates caused urinary tract infections in 59 cases (57.3%), wound infections in 21 cases (20.4%), sepsis in 15 cases (14.6%), pneumonia in 6 cases (5.8%), and abscesses in 2 cases (1.9%) (Fig. 3B). As shown in Fig. 3A, the isolates of cluster I were most common in patients 65 years old (75.7% versus 47.9%; P = 0.0014; OR = 3.4, 95% CI = 1.6 to 6.9). They were also more frequently implicated in urinary tract infections (57.3% versus 35.4%; P = 0.0147; OR = 2.4, 95% CI = 1.2 to 4.9) than other isolates that were resistant to cefotaxime and ceftazidime (Fig. 3B).
FIG. 3. Comparison of patient age (A) and clinical diagnostics (B) between E. coli isolates belonging to cluster I and other isolates resistant to cefotaxime and ceftazidime. (1), P = 0.001; (2), P = 0.01.
Cluster I isolates were also significantly more resistant to ciprofloxacin, gentamicin, tobramycin, amoxicillin-clavulanic acid, and piperacillin-tazobactam than other E. coli isolates (Table 3). However, these isolates exhibited a lower resistance to cefoxitin than did other strains (Table 3). Multiresistance pattern A was the most common pattern among the cluster I isolates (46.6%), followed by pattern D (9.7%) (Table 2).
A total of 67 of the 103 cluster I strains (65%) were collected during 2004 by two of the participating hospitals. Of these strains, 16 (23.9%) were isolated between January and June, and 51 (76.1%) were isolated from July to December (Fig. 1).
Isoelectric focusing. Seventy-three E. coli isolates, which included a representative sample of the different clusters identified by PFGE and of the unrelated strains, were tested by isoelectric focusing. Of the 73 isolates, 50 (68.5%) contained four -lactamases with apparent pI values of 5.4, 6.8, 7.4, and 8.6. The 41 cluster I isolates tested had this profile, while only 10 (31.2%) of the 32 isolates that belonged to other clusters had this profile (P < 0.0001; OR = 177.9, 95% CI = 9.9 to 3,180).
Molecular analysis of ESBLs. All 151 E. coli isolates resistant to cefotaxime and ceftazidime were screened with specific CTX-M-10-group primers. DNA from a total of 140 isolates was amplified; 133 isolates were identified as containing CTX-M-15, and 7 were identified as containing CTX-M-32. The remaining 11 strains were analyzed by using universal CTX-M-type primers; 5 strains contained CTX-M-14, and 6 did not amplify.
All isolates of the four clusters, as well as 8 (30.8%) of the 26 unrelated isolates, expressed CTX-M-15. All of the E. coli isolates expressing CTX-M-14 or CTX-M-32 exhibited individual PGFE profiles.
Sequence analysis of PCR products obtained with blaTEM- and blaOXA-1-specific primers identified the -lactamases TEM-1 (pI = 5.4) and OXA-30 (pI = 7.4). No PCR products were obtained by using the blaSHV, blaOXA-2, and blaOXA-10 primers. The band corresponding to the pI of 6.8 did not amplify with any of the specific primers for the following -lactamase groups: TEM, SHV, CTX-M, CTX-M-10, OXA-1, OXA-2, OXA-9, OXA-10, OXA-22, OXA-23, OXA-51, and GES-1 (http://www.lahey.org/studies/webt.asp).
Insertion sequences. Eighteen isolates from clusters I, II, III, or IV, which expressed CTX-M-15 were analyzed by using PROM+ and PRECTX-M-3B primers, as described in Materials and Methods. Cluster I, III, and IV strains yielded PCR products of approximately 1,000 bp. Further sequencing of this fragment revealed that blaCTX-M-15 was directly linked to an upstream ISEcp1-like element, previously implicated in expression and mobilization of CTX-M-15 (13). These isolates failed to yield amplicons using specific primers designed to amplify a 400-bp fragment spanning the IS26 element and blaCTX-M-15—a characteristic of the epidemic strain A from the United Kingdom (32). In contrast, 8 of the 11 isolates belonging to cluster II yielded amplicons of approximately 2,000 bp and contained the fragment spanning the IS26 element and blaCTX-M-15 characteristic of strain A (32).
Identification of additional resistance genes. The tetA gene was detected in 10 E. coli isolates resistant to tetracycline. Resistance to trimethoprim and to sulfamides was associated with a combination of sul2 and dfrA14 (eight isolates) or a combination of sul1 and dfrA17 (two isolates). In relation to the aminoglycosidase resistance genes, seven isolates resistant to both gentamicin and tobramycin had aac(6')Ib and aac(3)IIb genes. The remaining three isolates, which were resistant to tobramycin but susceptible to gentamicin, contained only aac(6')Ib.
The acrR gene was amplified in all 10 isolates examined. Six of these isolates exhibited no gene modifications. In contrast, two exhibited a single amino acid substitution (Gly28Val), and two exhibited a single nucleotide deletion (Gua85 and Cyt249).
Virulence factors. All of the 27 isolates examined harbored the same three virulence genes with one exception. Two genes encoded siderophores (iutA and fyuA), and one encoded a serum survival factor (traT). One isolate had only the traT gene. None of the genes encoding S, G, M, and F1C fimbriae were detected in these 27 isolates.
DISCUSSION
We have previously described significant increases in ESBL production, by both nosocomial and community-acquired E. coli in recent years in Spain (21). Other worldwide studies have documented similar findings (6, 30). This increase has been attributed to the rising prevalence of the CTX-M family of ESBLs that has emerged as an important and rapidly developing problem worldwide (1, 9, 25). The first CTX-M-type enzyme detected in Spain was CTX-M-9, which was reported in 1996 (26). In 2003, the first report documenting the isolation of a CTX-M-like ESBL in the United States was published (nine strains from five U.S. states) (27).
At present, the more prevalent CTX-M-types isolated from clinical samples in Spain have been CTX-M-9 and the genetically related CTX-M-14, followed by CTX-M-10 (9, 17, 25). No clonal dissemination of ESBL-producing E. coli was observed in a nationwide study of 40 Spanish centers in 2000 (9). CTX-M-15 was not detected in any of the three clinical Spanish studies mentioned (9, 17, 25). Also, CTX-M-15 was not detected in one study of fecal carriers performed in Madrid in 2003 (29). All ESBLs found were of the CTX-M-9, CTX-M-10, and CTX-M-14 types (29). However, in another fecal carrier study from Barcelona (2001 and 2002), CTX-M-15 was detected in five cases, as were both CTX-M-9 and CTX-M-14 (17).
The data reported here indicate that dissemination of high-level cefotaxime- and ceftazidime-resistant E. coli may be attributable to the following. First, the majority of resistant isolates were clonally associated and may have caused epidemics in several clinical settings. Second, ESBL CTX-M-15 demonstrates the ability to spread among different clusters of E. coli. Third, the simultaneous presence of other ESBLs of the CTX-M type, such as CTX-M-32 and CTX-M-14, may produce the same broad-spectrum cephalosporin resistance profile.
The main epidemic E. coli strain was detected in 2004 and 2005 in isolates from patients of very distinct origins, including three hospitals with a joint catchment population of approximately 1,300,000 persons (total population of the Madrid Autonomous region is approximately 5.5 million) covering several distinct administrative health areas, eight different nursing homes, and one community healthcare center. Infection control measures were reinforced afterward.
ESBL CTX-M-15 was first described in New Delhi in 1999 and is carried on large plasmids in E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes (13). Since then, outbreaks of CTX-M-15 E. coli producers have been described in France, the United Kingdom, and Canada (2, 15, 32). In some studies, the simultaneous presence of OXA-30 was also reported (7).
The great majority of isolates belonging to our epidemic cluster I were from urinary tract infections that affect older patients, a finding in accordance with other studies (15, 19). However, our data may indicate a complex underlying epidemiology of these CTX-M-15 producers. The high spreading capacity of CTX-M-15 includes two possible scenarios. One is the spread of an epidemic clone with some selective advantages (e.g., multiple antibiotic resistance and enhanced virulence) between different hospitals, long-term care facilities, and the community; the other is the horizontal transfer of plasmids or genes that carry blaCTX-M-15 alleles. Here we demonstrate the presence of blaCTX-M-15 within two different genetic environments in the same geographical region. The blaCTX-M-15 gene was directly linked to an upstream insertion sequence ISEcp1-like element (clusters I, III, and IV of the present study), as described in Canada, Cameroon, and India (2, 7, 13). In other isolates (eight strains of cluster II of the present study), an IS26 element was inserted within ISEcp1-like element preceding the blaCTX-M-15 gene, as described for epidemic strain A in the United Kingdom (32).
In addition to -lactamase resistance mechanisms, CTX-M-15 E. coli producers carried an important pool of mobile resistance genes including tetA, dfrA14 or dfrA17, sul1 or sul2, aac(6')Ib, and aac(3)IIb. Resistance to gentamicin and tobramycin was associated with a combination of the aac(6')Ib and aac(3)IIb genes, while resistance to tobramycin was linked to aac(6')Ib.
We also examined possible mutations in acrR, the regulator of the acrAB gene encoding a multidrug efflux pump that has been associated with antimicrobial resistance—principally fluoroquinolone resistance (31). Four of the seven multiresistant E. coli isolates tested demonstrated different amino acid substitutions or deletions in the acrR system; two of them had an amino acid substitution in a position (Gly28) previously described to be connected to norfloxacin, chloramphenicol, and tetracycline resistance (31). The acquisition of antibiotic resistance may be associated with decreased expression of virulence determinants (12). This has also been observed in ESBL E. coli producers isolated from urinary tract infections (15). We did not detect F1C, S, G, and M fimbria genes by PCR in our strains (that came from noninvasive infections in the 86% of the cases); however, the strains isolated from blood also had the same virulence genetic profile.
In the present study, 35% of the cluster I isolates were implicated in community-acquired infections. With probable origins of hospitals or nursing homes, these findings are demonstrative of the disruption of hospital-community barriers. In addition, the high and increasing use of fluoroquinolones in Spain and other countries (21) may be associated with a coselection phenomenon, which facilitates the persistence and spread of this epidemic strain in fecal flora of healthy carriers. Long-term care centers may represent a significant reservoir for multiresistant ESBL-producing E. coli isolates, and infection control efforts must be addressed in these settings.
In summary, the present study reports the spread of epidemic resistant E. coli isolates between the community, long-term care facilities and hospital settings. It also documents the first outbreak of a CTX-M-15 E. coli producer in Spain, affecting patients admitted to hospitals, nursing homes, and community outpatient centers. The CTX-M-15 extended-spectrum -lactamase was found to be widespread in a main epidemic E. coli strain and also in the majority of other isolates resistant to cefotaxime and ceftazidime. The same extended-spectrum cephalosporin resistance profile was also due to isolates expressing other ESBLs such as CTX-M-32 and CTX-M-14. The majority of these isolates were found to be susceptible to carbapenems but resistant to the vast majority of antibiotics tested. Broad dissemination of high-level cefotaxime- and ceftazidime-resistant E. coli raises important clinical and epidemiological concerns.
ACKNOWLEDGMENTS
This study was supported by research grants from the Instituto de Salud Carlos III, Programa Intramural (reference 03/ESP32); the Fondo de Investigaciones Sanitarias, Ministry of Health, Madrid, Spain (FIS PI040837); and the Red Espaola de Investigacion en Patología Infecciosa (REIPI). J.O. was supported by the Direccion General de Salud Pública, Ministry of Health, Madrid, Spain (reference SBVI1284/02-13).
FOOTNOTES
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Microbiology Department, Hospital Gregorio Maraon, Madrid, Spain
Microbiology Department, Fundacion Hospital de Alcorcon, Alcorcon, Madrid, Spain
Microbiology Department, Hospital Severo Ochoa, Leganes, Madrid, Spain
Microbiology Department, Centro de Especialidades Argüelles, Hospital Puerta de Hierro, Madrid, Spain
Consejo Superior de Investigaciones Científicas, Madrid, Spain
ABSTRACT
A total of 151 Escherichia coli strains resistant to cefotaxime and ceftazidime were isolated during a prospective surveillance study. These strains were characterized by clinical, microbiological, and molecular analyses and were distributed into four clusters of 103, 11, 6, and 5 isolates, along with 25 unrelated strains. The principal cluster was isolated from urine, wound, blood, and other samples in three hospitals, eight nursing homes, and a community healthcare center. This cluster was associated with both nosocomial (65%) and community-acquired (35%) infections. Most strains were resistant to ciprofloxacin, gentamicin, tobramycin, cefepime, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole but were susceptible to imipenem. All isolates from the four clusters expressed the extended-spectrum -lactamase (ESBL) CTX-M-15. This enzyme was also present in 8 (30.8%) of the 26 unrelated isolates. The other ESBLs, CTX-M-14 and CTX-M-32, were detected in five and seven cases, respectively, but they were detected in individual E. coli isolates only. In three clusters, blaCTX-M-15 alleles were linked to an ISEcp1-like element, while in eight strains of cluster II an IS26 element preceded the blaCTX-M-15 allele. An additional pool of resistance genes included tetA, drfA14 or dfrA17, sul1 or sul2, aac(6')Ib, and aac(3)IIb. All except one of the 27 isolates tested for genetic virulence markers harbored the same three virulence genes: iutA and fyuA (siderophores), and traT (serum survival factor). Epidemic or occasional isolates of cefotaxime- and ceftazidime-resistant E. coli can spread between distinct health facilities including hospitals, community health centers, and long-term care centers.
INTRODUCTION
The production of extended spectrum -lactamases (ESBLs) is one of the major sources of resistance to extended-spectrum cephalosporins in Enterobacteriaceae (16). Classically, plasmid-mediated ESBL enzymes have been of the TEM and SHV types, but in recent years CTX-M-type ESBLs have been increasingly found throughout the world (1). CTX-M enzymes predominantly hydrolyze cefotaxime but are weakly active against ceftazidime. However, some ESBLs of the CTX-M family display increased hydrolytic activities against ceftazidime, as is the case for CTX-M-15 (24) and CTX-M-32 (4).
In recent years, ESBL production in Enterobacteriaceae, particularly Escherichia coli, has significantly increased in several countries, including Spain (6, 21, 30). This increase is primarily due to the spread of CTX-M-type ESBLs (25).
ESBL dissemination in E. coli is usually due to plasmid transmission between unrelated strains (9), while clonal spreading is more frequent in other Enterobacteriaceae, such as Klebsiella pneumoniae (23). However, outbreaks of ESBL-producing E. coli clones have recently been described (2, 15, 18, 19, 32). In Spain, the proportion of E. coli CTX-M producers is rapidly increasing in both nosocomial and community-acquired infections (21, 25).
We have observed an apparent and alarming increase of cefotaxime- and ceftazidime-resistant strains among the E. coli isolates submitted to our laboratory. These events prompted the present study in which we describe the emergence and spread of high-level cefotaxime- and ceftazidime-resistant E. coli isolates, their complex molecular epidemiology, and the characterization of several clusters of multidrug-resistant CTX-M-15-producing E. coli isolates. Epidemic strains were detected in patients admitted to hospitals and nursing homes for the elderly, as well as in patients attending an outpatient community healthcare center.
MATERIALS AND METHODS
Study design. A prospective surveillance study of infections caused by ESBL-producing E. coli is currently ongoing in the Autonomous Community of Madrid (Spain). Three hospitals located in three separate geographic areas participated in the study and included Hospital Gregorio Maraon (estimated catchment population of 650,000), Hospital Fundacion de Alcorcon (estimated catchment population of 250,000, including eight associated nursing homes), Hospital Severo Ochoa de Leganes (estimated catchment population of 380,000), and a community healthcare center, Centro de Especialidades Argüelles. All collected the initial E. coli isolates that displayed ESBL production and sent them to the Centro Nacional de Microbiología, a public health reference institution, to confirm ESBL production and to study the molecular epidemiology of the isolates and the ESBL type. Here we describe the analysis of all ESBL-producing E. coli strains, isolated between January 2004 and August 2005, which exhibited resistance to cefotaxime (>32 μg/ml) and ceftazidime (16 μg/ml).
Infections and patient characteristics. The participating institutions collected the following information: personal patient data (code, age, sex, clinical diagnosis, and risk factors), hospital and departmental data, and isolate data (clinical sample and antimicrobial susceptibility). This information was entered into the Whonet software program, a free microbiological database (WHO Collaborating Center for the Surveillance of Antibiotic Resistance).
Patients with acquired community infections were those presenting at community health care centers and those who had a positive culture at the time of or within 48 h of hospitalization; in both cases, patients had no previous contacts with hospitals or long-term care facilities in the last 2 weeks.
Antimicrobial susceptibility testing and ESBL production detection. E. coli isolates were identified and antibiotic susceptibility was tested according to standard microbiological procedures performed in each clinical laboratory. At the Centro Nacional de Microbiología, isolates were subcultured in both Columbia blood agar and MacConkey agar to ensure viability and purity. The identification of all isolates was confirmed according to standard microbiological methods; susceptibility testing was initially carried out by microdilution (BD Phoenix Automated Microbiology System; Becton Dickinson Diagnosis, Sparks, MD) and the disk diffusion reference method (5). The antimicrobial agents tested were as follows: ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefoxitin, cefotaxime, ceftazidime, cefepime, amikacin, gentamicin, tobramicin, ciprofloxacin, imipenem, and trimethoprim-sulfamethoxazole. ESBL production was confirmed as described by the Clinical Laboratory Standards Institute (5); inhibition zones obtained by using disks that contained cefotaxime (30 μg) and ceftazidime (30 μg) were compared to those containing cefotaxime-clavulanic acid (30 and 10 μg) and ceftazidime-clavulanic acid (30 and 10 μg) (Oxoid, Madrid, Spain), respectively. In addition, the cefotaxime and cefotaxime-clavulanic acid MICs, as well as for ceftazidime and ceftazidime-clavulanic acid, were determined by the E-test method (AB-Biodisk, Solna, Sweden). Antimicrobial susceptibility results were interpreted according to the breakpoints recommended by the Clinical Laboratory Standards Institute (5). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains.
Molecular epidemiology. The molecular epidemiology of E. coli isolates that were resistant to cefotaxime and ceftazidime was determined by pulsed-field gel electrophoresis (PFGE). Total bacterial DNA was digested with XbaI (MBI Fermentas, Vilnius, Lithuania), and DNA fragments were separated on a 1% agarose gel in 0.5x Tris-borate-EDTA buffer by using the CHEF Mapper apparatus (Bio-Rad, Madrid, Spain). The conditions were as follows: 14°C, 6 V/cm, pulse of 2 to 54 s, and 24 h. Gels were stained with ethidium bromide and photographed under UV light. Genetic similarity was calculated by the unweighted pair group method using arithmetic averages and presented in a dendrogram. Similarity was calculated by using Dice coefficients with a tolerance of 1.8% (Fingerprinting II Software; Bio-Rad). Well-resolved bands that corresponded to fragments exceeding 48.5 kb were included in the computer analysis.
Isoelectric focusing. Isoelectric focusing was carried out on a total of 73 E. coli isolates representing the major clones identified by PFGE. Bacteria, exponentially growing at 37°C in Luria-Bertani medium, were harvested, and cell-free lysates were prepared by sonication. Isoelectric focusing was performed by applying this crude extract to Phast gels (pH = 3 to 9) in a Phastsystem apparatus (Pharmacia AB, Uppsala, Sweden) (10). -Lactamases with known pI values of 5.9, 5.4, 7.6, and 8.1 were used in parallel as controls. Gels were stained with 500 μg of nitrocefin (Oxoid)/ml to identify -lactamase bands.
Molecular analysis of ESBLs. Based on both antibiotic resistance profiles and pI values, molecular identification of -lactamases was carried out by PCR amplification and DNA sequencing. All -lactamases detected by isoelectric focusing were characterized by molecular methods. Genomic DNA from wild-type isolates was used as a template for PCR. ESBL amplification was performed with the appropriate primers and cycling conditions for the TEM, SHV, CTX-M, OXA-1, OXA-2, and OXA-10 -lactamase groups, as described elsewhere (3, 22, 28). The following primers were designed to amplify and sequence the blaCTX-M-15 gene under standard conditions (CTX-M-15-F, 5'-GTT AAA AAA TCA CTG CG-3'; CTX-M-15-R, 5'-CAA ACC GTT GGT GAC G-3'). PCR products were separated on 0.8% agarose gels, stained with ethidium bromide, visualized under UV light, further purified by using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany), and sequenced by using an ABI Prism 377 automated sequencer (Perkin-Elmer, Norwalk, CT).
Insertion sequences. Linkage of blaCTX-M-15 alleles with insertion sequences (IS) was investigated; ISEcp1-like elements, previously implicated in their expression and mobilization (13), were confirmed with the primers PROM+ and PRECTX-M-3B as described previously (24, 32). In addition, specific primers were used to amplify a 400-bp fragment spanning the link between IS26 (inserted between ISEcp1 and blaCTX-M-15) and blaCTX-M-15; this fragment is believed to be characteristic of the epidemic E. coli strain A, a CTX-M-15 producer from the United Kingdom (32).
Identification of additional resistance genes. Based on antibiotic resistance profiles, additional mechanisms of antibiotic resistance were studied in 10 multiresistant CTX-M-15-producing isolates. Molecular identification of sul1, sul2, tetA, aac(6')Ib, aac(3)IIb, dfrA14, and dfrA17 genes was carried out by PCR amplification and DNA sequencing with primers previously described (8, 14, 15, 20). In addition, the acrR gene of the AcrAB efflux system was amplified, including the promoter-operator region, as described elsewhere (31).
Virulence factors. A total of 27 isolates from different clinical samples belonging to cluster I (12 from urine, 9 from blood, 5 from wounds, and 1 from the respiratory tract) and from different geographical sources (15 from three hospitals, 8 from the community, and 4 from four long-term care facilities) were evaluated for the presence of 10 genes encoding putative virulence factors characteristic of extraintestinal pathogenic E. coli using PCR as described previously (11). These genes included those encoding S fimbriae (sfaS), F1C fimbriae (focG), M blood group antigen-specific M fimbriae (bma), glucosaminyl-specific G fimbriae (gaf), Dr family adhesins (afa/dra), toxin associated with extraintestinal pathogenic E. coli (cnf1), siderophores as aerobactin (iutA), yersiniabactin (fyuA), the serum survival gene (traT), and the invasion of brain endothelium gene (ibeA).
Statistical analyses. Differences in the prevalence of antibiotic resistance between different groups were assessed by using the Fisher exact test. Association was determined by calculation of the odds ratio (OR) with 95% confidence intervals (CI). The null hypothesis was rejected for P values of <0.05. Statistical analyses were performed by using GraphPad Prism version 3.02 software (GraphPad Software, Inc., San Diego, CA).
RESULTS
Clinical isolates and population study. From January 2004 to August 2005, a total of 525 unduplicated E. coli ESBL producers were collected. Of these isolates, 151 (28.8%) were simultaneously resistant to cefotaxime and ceftazidime. A total of 65 (43%) were from males, 84 (55.6%) were from females, and 2 were from patients whose the gender was not reported. A total of 15 (9.9%) were from children 14 years of age, 21 (13.9%) were from 15- to 64-year-old patients, and 101 (66.9%) were from patients of 65 years; in 14 cases, the patient's age was unknown.
Of the 151 E. coli isolates, 76 (50.3%) were isolated from the urinary tract, 30 (19.9%) were from wounds, and 22 (14.6%) were from blood; the remaining 23 cases were isolated from other clinical samples (15.2%). Ninety-eight of the patients (64.9%) were admitted into different hospital departments: 33 (21.8%) to internal medicine, 16 (10.6%) to the emergency room, 12 (7.9%) to pediatric units, 7 (4.6%) to surgery, 5 (3.3%) to the intensive care unit, and 25 (16.5%) to other departments. Twenty-six patients (17.2%) came from long-term care facilities for elderly people, and twenty-one patients (13.9%) came from the community. In six cases, this information was not available.
In total, 92 isolates (60.9%) produced nosocomial infections (including those from nursing homes), 55 (36.4%) produced community-acquired infections, and for 4 isolates this information is unknown.
The clinical diagnoses included urinary tract infections (76 cases or 50.3%), wound infection (30 cases [19.9%]), sepsis (22 cases [14.6%]), pneumonia (7 cases [4.6%]), peritonitis (3 cases [2%]), and abscess (2 cases [1.3%]).
Seventy-nine patients (52.3%) had predisposing underlying conditions, with twenty-one of these being respiratory and/or cardiac disease (13.9%). Also among these conditions were 16 cases of impaired immunity (10.6%) (diabetes, 6; tumoral pathology, 4; transplantation, 3; human immunodeficiency virus infection, 2; rheumatic arthritis, 1), 11 cases of cognitive disorders (7.3%), 7 cases of urinary diseases (4.6%) (including 3 with a vesical catheter), 6 cases of premature birth (4%), and 4 cases of liver pathology (2.6%).
FIG. 1. Monthly distribution of total cases of E. coli isolates belonging to cluster I and other isolates resistant to cefotaxime and ceftazidime in two participating hospitals during 2004.
Antimicrobial susceptibility. Data on antimicrobial susceptibility for all 151 E. coli isolates resistant to cefotaxime and ceftazidime are provided in Table 1. The prevalences of antimicrobial resistance, calculated according to the MICs, were as follows: 59.9% for amoxicillin-clavulanic acid, 38.8% for piperacillin-tazobactam, 98% for cefepime, 8% for cefoxitin, 89.3% for ciprofloxacin, 82% for cotrimoxazole, 70% for gentamicin, 82.7% for tobramycin, and 1.3% for amikacin (Table 1).
Compared to nosocomial infections, community-acquired infections exhibited a lower resistance to gentamicin (56.4% versus 71%; P = 0.001; OR = 0.38, 95% CI = 0.18 to 0.79) and tobramycin (72.2% versus 87.6%; P = 0.025; OR = 0.36, 95% CI = 0.15 to 0.86). Most of the isolates were resistant to the majority of antibiotics tested. As shown in Table 2, 56 of the isolates (37.1%) exhibited multiresistance pattern A, being susceptible to amikacin, cefoxitin, and imipenem. Eleven of the isolates (7.3%) were classified as having multiresistance pattern B, being susceptible to amikacin and imipenem. Eleven of the isolates (7.3%) were susceptible to amikacin, cefoxitin, imipenem, and gentamicin (pattern C).
Molecular epidemiology. Cluster analyses of DNA fingerprinting performed on the 151 isolates is shown in Fig. 2. XbaI did not digest the DNA of one isolate.
FIG. 2. Dendrogram that illustrates the genetic relationship of 150 ESBL-producing E. coli isolates resistant to cefotaxime and ceftazidime. , isolate identification number.
Using PFGE analysis, four clusters of isolates were detected that exhibited a genetic relatedness of 85 to 100% (Fig. 2). A total of 103 isolates made up cluster I, 11 isolates made up cluster II, 6 isolates made up cluster III, and 5 isolates made up cluster IV. The remaining 25 isolates had a genetic similarity of <85% (Fig. 2) and were considered unrelated. Cluster I was distributed among all participant centers and included nosocomial (65%) and community (35%) isolates.
Clusters II and IV were distributed among three of the participant centers, whereas the six isolates of cluster III belonged exclusively to one of the centers.
Of the cluster I isolates, 3 (2.9%) were from children of 14 years of age, 13 (12.6%) were from 15- to 64-year-old patients, and 78 (75.7%) were from patients of 65 years; in nine cases, the patient's age was unknown (Fig. 3A). These isolates caused urinary tract infections in 59 cases (57.3%), wound infections in 21 cases (20.4%), sepsis in 15 cases (14.6%), pneumonia in 6 cases (5.8%), and abscesses in 2 cases (1.9%) (Fig. 3B). As shown in Fig. 3A, the isolates of cluster I were most common in patients 65 years old (75.7% versus 47.9%; P = 0.0014; OR = 3.4, 95% CI = 1.6 to 6.9). They were also more frequently implicated in urinary tract infections (57.3% versus 35.4%; P = 0.0147; OR = 2.4, 95% CI = 1.2 to 4.9) than other isolates that were resistant to cefotaxime and ceftazidime (Fig. 3B).
FIG. 3. Comparison of patient age (A) and clinical diagnostics (B) between E. coli isolates belonging to cluster I and other isolates resistant to cefotaxime and ceftazidime. (1), P = 0.001; (2), P = 0.01.
Cluster I isolates were also significantly more resistant to ciprofloxacin, gentamicin, tobramycin, amoxicillin-clavulanic acid, and piperacillin-tazobactam than other E. coli isolates (Table 3). However, these isolates exhibited a lower resistance to cefoxitin than did other strains (Table 3). Multiresistance pattern A was the most common pattern among the cluster I isolates (46.6%), followed by pattern D (9.7%) (Table 2).
A total of 67 of the 103 cluster I strains (65%) were collected during 2004 by two of the participating hospitals. Of these strains, 16 (23.9%) were isolated between January and June, and 51 (76.1%) were isolated from July to December (Fig. 1).
Isoelectric focusing. Seventy-three E. coli isolates, which included a representative sample of the different clusters identified by PFGE and of the unrelated strains, were tested by isoelectric focusing. Of the 73 isolates, 50 (68.5%) contained four -lactamases with apparent pI values of 5.4, 6.8, 7.4, and 8.6. The 41 cluster I isolates tested had this profile, while only 10 (31.2%) of the 32 isolates that belonged to other clusters had this profile (P < 0.0001; OR = 177.9, 95% CI = 9.9 to 3,180).
Molecular analysis of ESBLs. All 151 E. coli isolates resistant to cefotaxime and ceftazidime were screened with specific CTX-M-10-group primers. DNA from a total of 140 isolates was amplified; 133 isolates were identified as containing CTX-M-15, and 7 were identified as containing CTX-M-32. The remaining 11 strains were analyzed by using universal CTX-M-type primers; 5 strains contained CTX-M-14, and 6 did not amplify.
All isolates of the four clusters, as well as 8 (30.8%) of the 26 unrelated isolates, expressed CTX-M-15. All of the E. coli isolates expressing CTX-M-14 or CTX-M-32 exhibited individual PGFE profiles.
Sequence analysis of PCR products obtained with blaTEM- and blaOXA-1-specific primers identified the -lactamases TEM-1 (pI = 5.4) and OXA-30 (pI = 7.4). No PCR products were obtained by using the blaSHV, blaOXA-2, and blaOXA-10 primers. The band corresponding to the pI of 6.8 did not amplify with any of the specific primers for the following -lactamase groups: TEM, SHV, CTX-M, CTX-M-10, OXA-1, OXA-2, OXA-9, OXA-10, OXA-22, OXA-23, OXA-51, and GES-1 (http://www.lahey.org/studies/webt.asp).
Insertion sequences. Eighteen isolates from clusters I, II, III, or IV, which expressed CTX-M-15 were analyzed by using PROM+ and PRECTX-M-3B primers, as described in Materials and Methods. Cluster I, III, and IV strains yielded PCR products of approximately 1,000 bp. Further sequencing of this fragment revealed that blaCTX-M-15 was directly linked to an upstream ISEcp1-like element, previously implicated in expression and mobilization of CTX-M-15 (13). These isolates failed to yield amplicons using specific primers designed to amplify a 400-bp fragment spanning the IS26 element and blaCTX-M-15—a characteristic of the epidemic strain A from the United Kingdom (32). In contrast, 8 of the 11 isolates belonging to cluster II yielded amplicons of approximately 2,000 bp and contained the fragment spanning the IS26 element and blaCTX-M-15 characteristic of strain A (32).
Identification of additional resistance genes. The tetA gene was detected in 10 E. coli isolates resistant to tetracycline. Resistance to trimethoprim and to sulfamides was associated with a combination of sul2 and dfrA14 (eight isolates) or a combination of sul1 and dfrA17 (two isolates). In relation to the aminoglycosidase resistance genes, seven isolates resistant to both gentamicin and tobramycin had aac(6')Ib and aac(3)IIb genes. The remaining three isolates, which were resistant to tobramycin but susceptible to gentamicin, contained only aac(6')Ib.
The acrR gene was amplified in all 10 isolates examined. Six of these isolates exhibited no gene modifications. In contrast, two exhibited a single amino acid substitution (Gly28Val), and two exhibited a single nucleotide deletion (Gua85 and Cyt249).
Virulence factors. All of the 27 isolates examined harbored the same three virulence genes with one exception. Two genes encoded siderophores (iutA and fyuA), and one encoded a serum survival factor (traT). One isolate had only the traT gene. None of the genes encoding S, G, M, and F1C fimbriae were detected in these 27 isolates.
DISCUSSION
We have previously described significant increases in ESBL production, by both nosocomial and community-acquired E. coli in recent years in Spain (21). Other worldwide studies have documented similar findings (6, 30). This increase has been attributed to the rising prevalence of the CTX-M family of ESBLs that has emerged as an important and rapidly developing problem worldwide (1, 9, 25). The first CTX-M-type enzyme detected in Spain was CTX-M-9, which was reported in 1996 (26). In 2003, the first report documenting the isolation of a CTX-M-like ESBL in the United States was published (nine strains from five U.S. states) (27).
At present, the more prevalent CTX-M-types isolated from clinical samples in Spain have been CTX-M-9 and the genetically related CTX-M-14, followed by CTX-M-10 (9, 17, 25). No clonal dissemination of ESBL-producing E. coli was observed in a nationwide study of 40 Spanish centers in 2000 (9). CTX-M-15 was not detected in any of the three clinical Spanish studies mentioned (9, 17, 25). Also, CTX-M-15 was not detected in one study of fecal carriers performed in Madrid in 2003 (29). All ESBLs found were of the CTX-M-9, CTX-M-10, and CTX-M-14 types (29). However, in another fecal carrier study from Barcelona (2001 and 2002), CTX-M-15 was detected in five cases, as were both CTX-M-9 and CTX-M-14 (17).
The data reported here indicate that dissemination of high-level cefotaxime- and ceftazidime-resistant E. coli may be attributable to the following. First, the majority of resistant isolates were clonally associated and may have caused epidemics in several clinical settings. Second, ESBL CTX-M-15 demonstrates the ability to spread among different clusters of E. coli. Third, the simultaneous presence of other ESBLs of the CTX-M type, such as CTX-M-32 and CTX-M-14, may produce the same broad-spectrum cephalosporin resistance profile.
The main epidemic E. coli strain was detected in 2004 and 2005 in isolates from patients of very distinct origins, including three hospitals with a joint catchment population of approximately 1,300,000 persons (total population of the Madrid Autonomous region is approximately 5.5 million) covering several distinct administrative health areas, eight different nursing homes, and one community healthcare center. Infection control measures were reinforced afterward.
ESBL CTX-M-15 was first described in New Delhi in 1999 and is carried on large plasmids in E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes (13). Since then, outbreaks of CTX-M-15 E. coli producers have been described in France, the United Kingdom, and Canada (2, 15, 32). In some studies, the simultaneous presence of OXA-30 was also reported (7).
The great majority of isolates belonging to our epidemic cluster I were from urinary tract infections that affect older patients, a finding in accordance with other studies (15, 19). However, our data may indicate a complex underlying epidemiology of these CTX-M-15 producers. The high spreading capacity of CTX-M-15 includes two possible scenarios. One is the spread of an epidemic clone with some selective advantages (e.g., multiple antibiotic resistance and enhanced virulence) between different hospitals, long-term care facilities, and the community; the other is the horizontal transfer of plasmids or genes that carry blaCTX-M-15 alleles. Here we demonstrate the presence of blaCTX-M-15 within two different genetic environments in the same geographical region. The blaCTX-M-15 gene was directly linked to an upstream insertion sequence ISEcp1-like element (clusters I, III, and IV of the present study), as described in Canada, Cameroon, and India (2, 7, 13). In other isolates (eight strains of cluster II of the present study), an IS26 element was inserted within ISEcp1-like element preceding the blaCTX-M-15 gene, as described for epidemic strain A in the United Kingdom (32).
In addition to -lactamase resistance mechanisms, CTX-M-15 E. coli producers carried an important pool of mobile resistance genes including tetA, dfrA14 or dfrA17, sul1 or sul2, aac(6')Ib, and aac(3)IIb. Resistance to gentamicin and tobramycin was associated with a combination of the aac(6')Ib and aac(3)IIb genes, while resistance to tobramycin was linked to aac(6')Ib.
We also examined possible mutations in acrR, the regulator of the acrAB gene encoding a multidrug efflux pump that has been associated with antimicrobial resistance—principally fluoroquinolone resistance (31). Four of the seven multiresistant E. coli isolates tested demonstrated different amino acid substitutions or deletions in the acrR system; two of them had an amino acid substitution in a position (Gly28) previously described to be connected to norfloxacin, chloramphenicol, and tetracycline resistance (31). The acquisition of antibiotic resistance may be associated with decreased expression of virulence determinants (12). This has also been observed in ESBL E. coli producers isolated from urinary tract infections (15). We did not detect F1C, S, G, and M fimbria genes by PCR in our strains (that came from noninvasive infections in the 86% of the cases); however, the strains isolated from blood also had the same virulence genetic profile.
In the present study, 35% of the cluster I isolates were implicated in community-acquired infections. With probable origins of hospitals or nursing homes, these findings are demonstrative of the disruption of hospital-community barriers. In addition, the high and increasing use of fluoroquinolones in Spain and other countries (21) may be associated with a coselection phenomenon, which facilitates the persistence and spread of this epidemic strain in fecal flora of healthy carriers. Long-term care centers may represent a significant reservoir for multiresistant ESBL-producing E. coli isolates, and infection control efforts must be addressed in these settings.
In summary, the present study reports the spread of epidemic resistant E. coli isolates between the community, long-term care facilities and hospital settings. It also documents the first outbreak of a CTX-M-15 E. coli producer in Spain, affecting patients admitted to hospitals, nursing homes, and community outpatient centers. The CTX-M-15 extended-spectrum -lactamase was found to be widespread in a main epidemic E. coli strain and also in the majority of other isolates resistant to cefotaxime and ceftazidime. The same extended-spectrum cephalosporin resistance profile was also due to isolates expressing other ESBLs such as CTX-M-32 and CTX-M-14. The majority of these isolates were found to be susceptible to carbapenems but resistant to the vast majority of antibiotics tested. Broad dissemination of high-level cefotaxime- and ceftazidime-resistant E. coli raises important clinical and epidemiological concerns.
ACKNOWLEDGMENTS
This study was supported by research grants from the Instituto de Salud Carlos III, Programa Intramural (reference 03/ESP32); the Fondo de Investigaciones Sanitarias, Ministry of Health, Madrid, Spain (FIS PI040837); and the Red Espaola de Investigacion en Patología Infecciosa (REIPI). J.O. was supported by the Direccion General de Salud Pública, Ministry of Health, Madrid, Spain (reference SBVI1284/02-13).
FOOTNOTES
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