JAC Advance Access originally published online on October 21, 2005
Journal of Antimicrobial Chemotherapy 2005 56(6):1152-1155; doi:10.1093/jac/dki395
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Surveillance of extended-spectrum ß-lactamases from clinical samples and faecal carriers in Barcelona, Spain
1 Servei de Microbiologia de l'Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain
* Corresponding author. Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Av. Sant Antoni M. Claret, 167, 08025 Barcelona, Spain. Tel: +34-932-919-071; Fax: +34-932-919-070; E-mail: fnavarror{at}santpau.es
Received 29 July 2005; returned 19 August 2005; revised 21 September 2005; accepted 3 October 2005
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Abstract |
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Objectives: The aim of the present study was to characterize and compare the extended-spectrum ß-lactamase (ESBL)-producing organisms isolated from clinical samples and faecal carriers in 2001 and 2002.
Methods: A total of 5251 Enterobacteriaceae isolated from clinical samples and 1321 stool samples were evaluated for the presence of ESBLs. The stool samples were spread onto plates of MacConkey agar containing 2 mg/L cefotaxime for selection of ESBL-producing strains. These strains were defined as those showing synergism between amoxicillin/clavulanic acid and third-generation cephalosporins. The ß-lactamases involved were characterized by isoelectric focusing, PCR assays and DNA sequencing.
Results: The prevalence of ESBL-producing strains among clinical Enterobacteriaceae was 1.7%. Of these, 87.6% produced CTX-M, 25.8% produced SHV and 2.2% were TEM-type-producing strains. All clinical ESBL-producing strains were Escherichia coli, with the exception of four Klebsiella pneumoniae and one Citrobacter freundii. The prevalence of faecal carriage of ESBL-producing organisms was 3.3%. Of these, 75% produced CTX-M-type enzymes followed by 22.7% SHV-producing strains. All faecal ESBL-producing strains were E. coli except for one Enterobacter cloacae and one Proteus mirabilis. This latter strain produced the PER-1 enzyme reported for the first time in Spain.
Conclusions: The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The different types of ESBLs found were similar in both contexts. The most prevalent ESBLs were the CTX-M-related enzymes, with nine different types, followed by SHV-12.
Keywords: ESBLs , CTX-M , SHV , PER-1
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Introduction |
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Enterobacteriaceae carrying extended-spectrum ß-lactamases (ESBLs) or plasmid-mediated cephamycinases have emerged as significant pathogens. Such strains, usually resistant to multiple antimicrobial agents, can be challenging to treat, as therapeutic options are few.1 Infections due to such strains are associated with prolonged hospital stays, increased healthcare costs and, in the setting of bloodstream involvement, increased mortality if appropriate therapy is delayed.1
Several papers have recently described the prevalence of ESBLs in the community, mainly in patients with urinary tract infections or ambulatory patients with chronic conditions.1–3 This marks a serious problem, as infections with ESBL-containing bacteria have mostly been described as nosocomially-acquired or nursing-home-related.1
Since 1994, a low prevalence of ESBL-producing strains has been observed at our hospital.4 This finding differs from the higher prevalence (3.3%) observed among faecal carriers between 2001 and 2002.5 At this point, the aim of the present study was to characterize and compare the ESBLs found in clinical and stool samples between 2001 and 2002.
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Materials and methods |
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Bacterial isolates
A total of 5251 Enterobacteriaceae strains, 3177 Escherichia coli, 361 Klebsiella pneumoniae, 113 Klebsiella oxytoca, 379 Proteus mirabilis, 17 Proteus vulgaris, 3 Proteus penneri, 106 Morganella morganii, 11 Providencia spp., 260 Enterobacter cloacae, 93 Enterobacter aerogenes, 84 Serratia marcescens, 67 Citrobacter freundii, 45 Citrobacter koseri, 498 Salmonella enterica and 37 Shigella spp., were isolated in 2001 and 2002. All strains were from non-duplicate clinically relevant samples of patients who attended the Hospital de la Santa Creu i Sant Pau and all of them were evaluated for the presence of ESBLs.
Forty-four ESBL-producing strains isolated from 1321 stool samples collected from patients who attended the emergency room from February to May 2001, from April to June 2002 and in October 20025 were also characterized.
Susceptibility test
Susceptibility to ß-lactam antibiotics was determined by the disc diffusion test on Mueller–Hinton agar (Maim, Vic, Spain), following NCCLS recommendations.6 The discs were purchased from Bio-Rad, Marnes-la-Coquette, France. The antibiotics tested were amoxicillin/clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, cefepime and imipenem. The MICs of cefotaxime and ceftazidime, with and without clavulanic acid, were later determined by Etest (AB Biodisk, Solna, Sweden). Strains producing ESBL were defined as strains showing synergism between amoxicillin/clavulanic acid and cefotaxime, ceftazidime, aztreonam or cefepime.
Isoelectric focusing
Isoelectric focusing was carried out as described previously.4 Enzyme activities in the revealed gel were detected by the iodometric method.
PCR assays
Specific primers for blaSHV, blaCTX-M-9-related and blaTEM were used as described previously.4 The following primers were respectively used for blaCTX-M-1-related, blaCTX-M-2-related, blaCTX-M-3-related and blaPER: CTX-M-1up (5'-AAGGCGTTTTGACAGACTAT-3') and CTX-M-1dn (5'-CCGTTTCCGCTATTACAA-3'), CTX-M-2up (5'-TAGGTGGTAATGGAGGAT-3') and CTX-M-2dn (5'-GTTCAGGAGCACATTTTTAA-3'), CTX-M-3up (5'-ATGGTTAAAAAATCACTGCG-3') and CTX-M-3dn (5'-CTATTACAAACCGTCGGTG-3'), and PER-1-A (5'-TGACGATCTGGAACCTTT-3') and PER-1-B (5'-AACTGCATAACTACTCC-3') in standard conditions.
DNA sequencing
DNA sequencing of PCR products was done by the dideoxy method with primers mentioned above, the Thermo SequenaseTM CyTM5 Dye Terminator Sequencing kit (Amersham Biosciences, Little Chalfont Buckinghamshire, UK) and the Automatic Laser Fluorescent DNA Sequencer (ALF, Amersham Biosciences, Uppsala, Sweden). Nucleotide and deduced protein sequences were analysed using the DNAstar Lasergene software package and using the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).
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Results |
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Eighty-nine clinically relevant ESBL-producing strains out of 5251 Enterobacteriaceae (1.7%) were isolated during 2001–2002 (Table 1). Of these, 84 were E. coli, 4 K. pneumoniae and 1 C. freundii.
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The blaCTX-M type was the most frequent ESBL, representing 87.6% out of 89 ESBL-producing strains (75 E. coli, 2 K. pneumoniae and 1 C. freundii). Of these, CTX-M-9 was the most frequent enzyme, found alone in 31 strains and associated with SHV-12 in 14. The second most frequent ESBL was CTX-M-14 with 23 strains. Finally, we also found seven CTX-M-1, one CTX-M-2, one CTX-M-3 and one CTX-M-32 (Table 1).
The SHV-type represented 25.8% of the 89 ESBL-producing strains detected (21 E. coli, and 2 K. pneumoniae). The main enzyme present was SHV-12, isolated alone in seven strains and associated with CTX-M-9 in 14. Thus, all strains with SHV-12 enzyme isolated in 2001 and 50% of these isolates in 2002 also produced the CTX-M-9 enzyme. Finally, we also found two SHV-2-producing strains.
The presence of TEM-type ESBLs was low, 2.2%; recovered from two E. coli strains (both in 2001), one carrying blaTEM-19 and one blaTEM-104.
A total of 44 ESBL-producing strains (including 42 E. coli, 1 P. mirabilis and 1 E. cloacae) from the 1321 stool samples were isolated (3.3%) (Table 1). We did not isolate any ESBL-producing K. pneumoniae during the three periods evaluated. No previous hospitalization was documented in faecal carriers in the 3 months prior to sample collection. From the 44 ESBL-producing strains, 33 produced a CTX-M enzyme (75%). Of these, 69.7% belonged to the CTX-M-9 group (18 CTX-M-9 and 5 CTX-M-14), whereas the remaining enzymes belonged to the CTX-M-1 group [one CTX-M-1, one CTX-M-3, four CTX-M-15, three CTX-M-29, and one the new CTX-M-34 enzyme (AY515297 [GenBank] )]. Apart from CTX-M types, the SHV-12 enzyme was detected in nine strains (20.4%). One strain expressed SHV-2 and another PER-1.
The CTX-M-34-carrying strain was isolated in April 2002 from a 27-year-old female. It was resistant only to nalidixic acid and remained susceptible to fluoroquinolones, aminoglycosides, tetracycline and chloramphenicol. The enzyme showed a pI near 8.2 and preference for cefotaxime substrate, like most of the CTX-M enzymes.
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Discussion |
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Although the prevalence of ESBL in our hospital is low, an increase in ESBL-producing clinical E. coli strains was observed between 1994 (0.08%) and 2003 (2.6%). This was due to an increase in the prevalence of CTX-M-related enzymes and the appearance of SHV-12. This situation has been also observed around Spain.2,7 The first CTX-M enzyme described in our hospital was the CTX-M-9 from an E. coli isolated in 1996;4 it was the most prevalent enzyme until 2003 when it was displaced by CTX-M-14. CTX-M-14 appeared in our hospital in 1999 and we observed a 3-fold increase between 2001 and 2002. Since 2001, we have also observed an increase in the diversity of CTX-M enzymes (CTX-M-1, CTX-M-2, CTX-M-3 and CTX-M-32).
The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The proportion of the different ESBL-types found in both environments was similar, with the CTX-M-type being the most prevalent enzyme found. We also emphasize the absence of the TEM-type enzymes in faecal samples, the first description to our knowledge of PER-1 enzyme in Spain in one P. mirabilis strain and the detection of the new enzyme CTX-M-34, not described in clinical samples to date. The rapid emergence of the CTX-M-type enzymes as the predominant ESBLs in faecal carriers is not an isolated phenomenon.2,3,5,7 Several elements, such as plasmids, insertion sequences, transposons or integrons, may be involved in the mobilization of CTX-M genes.3,7 These elements can also carry genes for resistance to multiple other antibiotics, including sulphonamides, trimethoprim, aminoglycosides, tetracycline and chloramphenicol. When we analysed the resistance to the other antimicrobial agents tested we observed that all the strains, except CTX-M-34-producing E. coli, showed multi-antibiotic resistance. CTX-M-9-, CTX-M-14- and SHV-12-producing strains were mostly resistant to streptomycin, sulphonamides, trimethoprim and nalidixic acid (data not shown).
One hypothesis concerning the possible diffusion of ESBLs among the community is through animal food products where the selection in commensal and in zoonotic enteropathogens for antimicrobial resistance is possible.8 Nevertheless, non-animal food stuffs could also contribute to this diffusion (ESBLs have been detected in animal food; data not shown). In a previous paper, we documented ESBL and plasmid-mediated cephamycinase-producing E. coli, probably transmitted within the community through the food supply.9 Moreover, other factors, such as mobile genetic elements like plasmids, transposons, integrons or bacteriophages, could also contribute.1,3,7,10
Although we do not have data on the previous administration of antibacterial drugs in all the studied patients, it is possible that healthy people could act as a reservoir of these ESBLs as a consequence of antibiotic use in the community. Further studies should be undertaken to determine the diffusion mechanisms of these enzymes, as well as the length of time an ESBL strain may be carried in the digestive tract.
Nucleotide sequence accession number
The accession number of the nucleotide sequence of the blaCTX-M-34 gene in the GenBank database is AY515297 [GenBank] .
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None to declare.
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Acknowledgements |
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This work was partially supported by grants FIS 97/1522 and FIS PI 020372 from the ‘Fondo de Investigaciones Sanitarias de la Seguridad Social de España’ and from ‘Red Española de Investigación en Patología Infecciosa’ (REIPI C03/14).
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References |
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1. Bradford PA. Extended-spectrum ß-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933–51.
2.
Rodriguez-Baño J, Navarro MD, Romero L et al. Epidemiology and clinical features of infections caused by extended-spectrum ß-lactamase-producing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: 1089–94.
3.
Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum ß-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–43.
4.
Sabaté M, Miró E, Navarro F et al. ß-Lactamases involved in resistance to broad-spectrum cephalosporins in Escherichia coli and Klebsiella spp. clinical isolates collected between 1994 and 1996, in Barcelona (Spain). J Antimicrob Chemother 2002; 49: 989–97.
5. Mirelis B, Navarro F, Miró E et al. Community transmission of extended-spectrum ß-lactamase. Emerg Infect Dis 2003; 9: 1024–5.[Web of Science][Medline]
6. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA, 2002.
7.
Valverde A, Coque TM, Sánchez-Moreno MP et al. Dramatic increase in prevalence of fecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain. J Clin Microb 2004; 42: 4769–75.
8.
Briñas L, Zarazaga M, Saenz Y et al. ß-Lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob Agents Chemother 2002; 46: 3156–63.
9. Prats G, Mirelis B, Miró E et al. Cephalosporin-resistant Escherichia coli among summer camp attendees with salmonellosis. Emerg Infect Dis 2003; 9: 1273–80.[Web of Science][Medline]
10. Muniesa M, Garcia A, Miró E et al. Bacteriophages and diffusion of ß-lactamase genes. Emerg Infect Dis 2004; 10: 1134–7.[Web of Science][Medline]
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