1 Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900; 2 Microbiology Laboratory, Ma'aynei-Hayeshua Hospital, Bnei Brak; 3 Ministry of Health Central Laboratories, Jerusalem, Israel; 4 Division of Microbiology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
Received 12 September 2004; returned 7 October 2004; revised 21 October 2004; accepted 28 October 2004
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Abstract |
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Methods: Extended-spectrum ß-lactamase (ESBL)-producing clinical isolates of E. coli from extra-intestinal sources were tested for susceptibility to non-ß-lactam drugs, and their serotypes were determined. Restriction enzyme digestion, followed by PFGE of DNA purified from isolates, was used to classify the phylogenetic relationship between them. Plasmid DNA from five isolates of different serotypes was used to transform an E. coli laboratory strain. The plasmids were partially sequenced.
Results: E. coli isolates from 86 patients, mostly elderly, were shown to be positive for inhibitor-susceptible ESBLs, and more resistant to cefotaxime than to ceftazidime. Ninety-six per cent of ESBL producers were also resistant to gentamicin, and 100% to trimethoprim/sulfamethoxazole and ciprofloxacin. All isolates belonged to one of five serotypes. PFGE analysis of purified DNA yielded 17 profiles. Sequencing of plasmids isolated from the transformants identified sul1, aac(6')-Ib and blaCTX-M-2. These genes were embedded in an integron, InS21.
Conclusions: Extra-intestinal infections with ESBL-producing E. coli of different serotypes and probably mixed clonality showed a surprising homogeneity in resistance profiles, with 100% being co-resistant to ciprofloxacin and trimethoprim/sulfamethoxazole, and 96% to gentamicin. Plasmid DNA from three isolates from different serotypes contained integron InS21, previously demonstrated in Salmonella enterica from Argentina. This is the first molecular identification of an ESBL gene and integron in Israel or neighbouring geographical areas.
Keywords: extended-spectrum ß-lactamases , Enterobacteriaceae , E. coli
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Introduction |
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The objective of this study was to examine the clonality of the outbreak and the molecular basis of the resistance.
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Materials and methods |
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During a period of 42 months (December 1997May 2001), we tested E. coli isolatesobtained from clinical extra-intestinal samplesfor ESBL production. Taxonomic determinations were performed by accepted biochemical tests and confirmed by Api20E (Analytab Products, Plainview, NY, USA). E. coli serological determination was performed according to classical methodology.7 Susceptibility to antibiotics was routinely tested by agar diffusion (discs from Oxoid Ltd, Basingstoke, UK) and results were evaluated according to the recommendations of the NCCLS (2003). The routine testing covered 16 different antibiotic agents, including expanded-spectrum cephalosporins. ESBL determination was performed as indicated in the Results section.
PFGE
DNA in agarose plugs containing 5 x 108 cfu lysed E. coli was digested with 5 U of SpeI/sample (New England Biolabs, UK) and fixed in slots of a 1% pulsed-field certified agarose gel (Life Technologies). PFGE was performed with a contour-clamped homogeneous electric field system (CHEF-DR III; Bio-Rad Laboratories, CA, USA) in 0.5 x TBE buffer [0.1 M Tris-HCl (pH 8.0), 0.1 M boric acid, 0.2 M EDTA].
Images of ethidium-bromide stained gels were analysed using BioNumerics software (Applied Maths, Belgium). E. coli isolates were considered to be in different groups if there was a difference of two or more bands in their PFGE pattern by computer comparison and visual inspection.
Identification of antibiotic resistance encoding genes and integron regions
Detection of antibiotic resistance encoding genes was performed using primers listed in Table 1. PCR was performed in 100 µL reaction mixtures, using 100 ng of plasmid DNA as template and 2.5 U of TaKaRa TAQ DNA polymerase (Takara Shuzo Co., Ltd, Shiga, Japan). PCR products were purified using a High Pure PCR Product Purification Kit (Roche, Mannheim, Germany). Nucleotide sequences were determined with an automated cycle-sequencing system on an ABI Prism 3100 Genetic Analyzer.
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Statistical significance was examined by the 2 test.
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Results |
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During 42 months, we isolated ESBL-producing E. coli from 96 extra-intestinal samples taken from 83 inpatients of the Department of Internal Medicine of our hospital, and we obtained four ESBL-producing isolates from three patients living in a nearby geriatric centre. E. coli susceptible to all examined oxyimino ß-lactam drugs (non-ESBLs) were obtained from 97 samples taken from 88 patients. Blood was the source of 18% of the ESBL-producing isolates and 55% of the ESBL-negative isolates. Urine was the source of 50% of the ESBL-producing isolates and 31% of the ESBL-negative isolates. Seventy-three per cent of patients with E. coli ESBL and 47% of those with non-ESBL E. coli were 85 years or older (P < 0.001), and 1% (one patient) with ESBL E. coli and 13% of those with non-ESBL E. coli were <65 years old (P < 0.001).
Resistance profiles of E. coli ESBL isolates
All isolates resistant to oxyimino-cephalosporins had surprisingly similar resistance profiles, and were considered ESBL positive on the basis of the following observations:
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Serotypes
The 100 ESBL-producing E. coli isolates from this study belonged to one of five serotypes (O101:H-, O153:H31, O102:H6, O2:H42 and a single isolate of O108:H31) (Table 2). None of these serotypes (combinations of O and H) was found among the non-ESBL strains. These serotypes (O:H types) have not been previously reported in human extra-intestinal infections. K1 and K5 antigens were not demonstrated in any of the isolates.
PFGE patterns
PFGE studies were performed on 54 isolates from 53 random patients. These isolates were subdivided into 17 genotypic profiles. As expected, none of the groups spanned more than one serotype. Some of the profiles, however, were more common than others. For example, a particular profile was found in 19 of 27 different isolates of serotype O153:H31, whereas seven profiles were unique for single isolates.
Antibiotic resistance of transformants
Plasmid DNA from five ESBL-producing isolates of the three most frequent serotypes (O101:H-, O153:H31 and O2:H42) was used to transform the E. coli laboratory strain XL1-Blue. Transformants selected on ampicillin agar (100 mg/L) were all ESBL (Table 2), and, in addition, resistant to gentamicin and sulfamethoxazole. All the transformant clones were susceptible to ciprofloxacin.
Molecular mapping of the plasmid
Plasmid DNA was used as template for PCR reactions with primers that can distinguish TEM, SHV, AmpC and CTX-M (see Table 1). CTX-M and TEM were successfully amplified from template DNA isolated from all the isolates. The CTX-M PCR products from the five isolates were sequenced and showed identity with the CTX-M-2 ESBL gene, and a 266 bp KLU upstream fragment found in most CTX-M-2 genes.8
We suspected that the CTX-M-2 might be located on an integron, due to the multiresistance phenotype we observed in the transformants. We used published primers to PCR amplify and sequence int1 using plasmid DNA as template isolated from DNA originating in the above three serotypes. We found the int1 gene in all transformants tested, indicating the presence of an integron on the tested plasmids.
Using additional primers (Table 1), we PCR-amplified and sequenced an entire integron on one of the isolated plasmids. It was identical to InS21,9 which contains sul1 and aac(6')-Ib. Sequencing of PCR products amplified from each of the other two isolates indicated that they too were identical to InS21, as we verified that all the genes in InS21 were present in each of the plasmids and that they were identically ordered.
TEM analysis
Primers specific for TEM (Table 1) were used to amplify and sequence this gene from the isolated plasmids of each of the donor strains. All showed 100% identity to TEM-1. Further sequencing identified the C-terminus of the Tn3 resolvase protein upstream of the TEM promoter sequence. Downstream of the TEM, a 142 bp region was found that is common in transposons, followed by 156 bp of the C-terminus (in the opposite orientation) of aacC2/aacC3 gentamicin-(3)-N-acetyl-transferase. This configuration of sequences was previously found only in the E. coli R-plasmid pWP116a.10
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Discussion |
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Sequencing showed the presence of an integron, InS21, incorporated into the plasmids isolated from clinical isolates of three different serotypes. This integron was previously isolated from Salmonella enterica.9 Besides the blaCTX-M-2, the integron carried sulfamethoxazole and an aminoglycoside resistance gene from the aac family. Outside the integron we identified a common TEM gene. The TEM with its upstream region may be part of a Tn3 transposon.
Since we were able to transfer most of the antibiotic resistances via transformation of plasmid DNA, and since plasmid DNA from isolates of different serotypes shared the same restriction endonuclease pattern, we conclude that the antibiotic resistance was incorporated into a number of distinct clones of E. coli, probably through horizontal transfer of plasmids. As antibiotic resistance genes were found in an integron, this may explain the broad resistance phenotype.
It is particularly alarming that the InS21 integron has appeared in plasmids harboured in E. coli, and that it has simultaneously been found in multiple serotypes not usually associated with UTIs. Although CTX-M-2-mediated ESBL has been reported in many countries during the last few years, the integron-mediated CTX-M-2 ESBL resistance has not been reporteduntil nowanywhere except Argentina.9
Our study raises important epidemiological questions. Serotyping and PFGE data indicate that the outbreak in our institution of E. coli ESBL infections involves several different clones. In addition, data from four additional hospitals in the Tel Aviv area indicate that this infection may be more widespread.5 Since the patients in both studies were elderly, it is likely that particular E. coli clones have adapted themselves to adhere to the urinary tract epithelia of geriatric patients or to catheters commonly used in elderly patients. Preliminary work in our laboratory has demonstrated a prevalence of biofilm-producers among the isolates of serotype O153:H31.
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Acknowledgements |
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We thank Abraham Nudelman (Department of Chemistry, Bar Ilan University) for helpful comments, Hagai Radotzki (pharmacist, Ma'aynei-Hayeshua Hospital) for drug use and pricing information, Sofia Volis (Laboratory of Microbiology, Ma'aynei-Hayeshua Hospital) and Ilana Slucky-Shraga (Department of Medicine, Ma'aynei-Hayeshua Hospital) for technical and medical support, Niels Frimodt-Møller (Statens Serum Institut) for helpful suggestions in all phases of this project and Flemming Scheutz (International Escherichia and Klebsiella Centre, World Health Organization, Copenhagen, Denmark) for help with the serotyping.
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Footnotes |
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* Contributed equally to this publication.
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References |
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