Enteropathogenic Escherichia coli Strains Carrying Genes Encoding the PER-2 and TEM-116 Extended-Spectrum -Lactamases Isolated from Children
http://www.100md.com
微生物临床杂志 2005年第6期
Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, 11600 Montevideo, Uruguay
Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina
Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain
ABSTRACT
We studied 13 extended-spectrum -lactamase (ESBL)-producing enteropathogenic Escherichia coli isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of blaPER-2 and blaTEM-116. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.
TEXT
The emergence of new resistance mechanisms is usually recognized when their corresponding genes reach hospital pathogens, by mobilization or capture of plasmids, transposons, or integrons. These resistance mechanisms are evidenced in the hospital environment only after sudden, and sometimes remarkable, changes in the resistance profile of nosocomial microorganisms. Therefore, it is difficult to define how the corresponding genes gain access to the hospital environment, as resistance is followed primarily in disease-causing microorganisms.
Even if commensally microorganisms play a role in disseminating resistance genes and represent a route for gaining access to pathogenic bacteria, they do not necessarily constitute their ultimate origin. A continuous relationship of those microorganisms with humans may complicate the evaluation of their contribution to resistance, as they may have been submitted to the same antibiotic pressure forces affecting pathogenic bacteria and thus may have gained resistance genes from other microorganisms. Moreover, even environmental microorganisms obtained from most sources may have been in previous contact with bacteria of human origin.
It is accepted that most plasmidic -lactamase-encoding genes (of prevalent broad and extended-spectrum -lactamases and recently carbapenemases) derive from chromosomally located genes of commensal or environmental microorganisms. However, most of these sources are not known, even for the most promiscuous genes, such as blaTEM. Kluyvera species, such as Kluyvera ascorbata, K. cryocrescens, and K. georgiana, have been suggested, for example, as reservoirs of one of the emergent groups of extended-spectrum -lactamases (ESBLs) worldwide: the CTX-M -lactamases (4, 6, 9, 18).
These enzymes appear to be natural ESBLs or "born oxyimino-cephalosporinases" (21), for whose expression there is no need for any mutational shift leading to a modification of the spectrum of activity. The only requisite is to find the right promoter, copy number, or even genetic environment, allowing the proper expression of the -lactamase-encoding gene, and therefore the expected resistance levels for an ESBL. Spread is due to their recruitment or mobilization to more promiscuous elements, such as plasmids harboring transposons or integrons.
Other worldwide prevalent ESBLs, especially TEM- and SHV-derived enzymes, emerge from mutations in genes coding for narrower-spectrum enzymes, already located in transferable elements, resulting in -lactamases with a wider hydrolytic activity (5). Surprisingly, TEM-derived ESBLs have been described only recently in this region (south of Brazil, Argentina, Paraguay, and Uruguay) (16). Additionally, PER-2, the second most prevalent ESBL (20) in isolates from Argentina, has never been reported in any other country.
Acute diarrheal disease is still an important cause of infectious morbidity in Uruguayan children (as well as in many other developing countries), enteropathogenic Escherichia coli (EPEC) being the most frequently found bacterial pathogen. It is usually associated with infants from the poorer social groups; the most prevalent serogroups are O111, O119, and O55 (in decreasing frequency) (23).
Antimicrobial treatment is not recommended for diarrheal diseases caused by enteropathogenic bacteria other than shigellae (i.e., EPEC). Therefore, antibiotic resistance genes harbored by these microorganisms can remain unnoticed and may be silently transferred between different bacterial species in the community or among patients.
We previously reported EPEC isolates resistant to several antibiotics, including 13 oxyimino-cephalosporin-resistant strains, from 68 EPEC strains originally isolated between October 1990 and April 1993. Some characteristics are shown in Table 1. They were obtained from diarrheal cultures of hospitalized children under 30 months old at one pediatric ward of Hospital de Nios "Pereira Rossell" in Montevideo, Uruguay (23).
We focused our attention on the 13 oxyimino-cephalosporin-resistant EPEC isolates, 11 of them belonging to serogroup O119 and the remaining 2 belonging to serogroup O142. Some of their features are displayed in Table 2. All strains were confirmed as eaeA and bfpA positive by PCR (7, 8). MICs, with and without clavulanic acid (4 μg/ml), of ceftriaxone (CRO), ceftazidime (CAZ), and cefepime (FEP) were determined as specified by the NCCLS (13) for isolates showing resistance to CAZ or cefotaxime in disk diffusion tests. Studies of EPEC strain susceptibility to a wide variety of antibiotics were performed by disk diffusion tests following NCCLS guidelines (14).
Antimicrobial susceptibility data suggested a more efficient hydrolysis of CAZ compared to CRO or FEP. Data on gentamicin and trimethoprim-sulfamethoxazole resistance were also compared, rendering three different antibiotypes, as shown in Table 2.
Crude extracts from late-log-phase cultures were obtained from all 13 oxyimino-cephalosporin-resistant EPEC isolates. ESBL characterization was performed by isoelectric focusing (IEF) in broad-range (pH 3 to 10) polyacrylamide gels as previously described (11). All crude extracts displayed only one -lactam-hydrolyzing band (pI 5.4) when revealed with an iodometric overlay system (19) using CRO (1,000 μg/ml) as the substrate.
Guided by IEF results, the presence of blaPER-2 (using primers PER-2F [5'-TGT GTT TTC ACC GCT TCT GCT CTG-3'] and PER-2R [5'-AGC TCA AAC TGA TAA GCC GCT TG-3']) and blaTEM genes (using primers OTR 3 [5'-ATG AGT ATT CAA CAT TTC CG-3'] and OTR 4 [5'-CCA ATG CTT AAT CAG TGA GG-3']) could also be inferred by PCR in each isolate. Plasmid DNA was extracted by alkaline lysis (3). The reaction mixture contained 2 μl of plasmid DNA, and 1.25 U of Taq DNA polymerase (Gibco/BRL) was added to a total volume of 50 μl containing 0.2 mM deoxynucleoside triphosphate, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 50 mM KCl, and 0.5 μM primers. The reaction protocol required an initial step of 5 min at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 20 min. PCR products were automatically sequenced in both strands by primer extension with the same primers, using an ABI PRISM 3700 DNA sequencer. The sequences showed strict identity with blaPER-2 (2) and blaTEM-116 (10).
Plasmid DNA was also used as the template in PCR screening for blaSHV and blaCTX-M using specific primers (1, 19), all results being negative.
PER-2 was first described by Bauernfeind et al. after being sequenced from an Argentinean Salmonella enterica serovar Typhimurium isolate (2). Other microorganisms harboring the same enzyme have been circulating in Buenos Aires since at least 1989; the enzyme was named, at that time, ARG-1 (A. Rossi, M. Quinteros, E. Couto, et al., Abstr. XI Congr. Latinoam. Microbiol., abstr. B49, 1991; M. A. Rossi, G. Gutkind, M. Quinteros, et al., Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 939, 1991). The gene was also recognized in other enterobacteria and related pathogens, including a 1993 Vibrio cholerae isolate (17, 20).
This study represents the first report of PER-2 in a microorganism outside Argentina (however, in a neighboring country, Uruguay).
In addition, we found that TEM-derived ESBLs were already present in Uruguay in the early 1990s. This is the second worldwide TEM-116 clinical report. This enzyme was reported very recently in Korea (10). An interesting coincidence in apparent pIs makes resolution of these two enzymes by IEF analysis very unlikely, perhaps masking one of them just by supposing, from previous data, the existence of the other in the region.
The first blaPER-2-blaTEM-116-carrying-EPEC isolate was obtained in November 1991 during a programmed survey. By the time the epidemiological work was concluded (April 1993) (23), two more groups of isolates carrying the same enzymes but showing different resistance profiles (3 and 1, respectively) were obtained (see details in Table 2). In all three profiles, the initial isolate corresponded to one child stool sample obtained at admission or within 24 h of admission. Other cultures were obtained from hospitalized patients and may correspond to dissemination of these EPEC strains in the ward. However, the three index case cultures are clear indicators of community circulation of the corresponding resistance genes before accessing the hospital. These cultures are probably part of the initial burst of plasmid-borne blaPER-2 diffusion that later involved many bacterial species.
Information about ESBLs in EPEC infections is not readily available. This can be due, at least in part, to the fact that laboratories usually do not devote resources to the identification of EPEC isolates and their antimicrobial susceptibility profile because antibiotic treatment is not indicated for EPEC diarrhea. Thus, data on the resistance of these strains is not routinely produced, as for other bacterial pathogens. Moreover, the attention of microbiologists usually focuses on pathogenic microorganisms but not on related bacteria of indigenous flora that may also develop resistance in treated patients under sustained selective pressure of misguided excessive antibiotic use.
Under these circumstances, EPEC strains are microbial pathogens that can cause in our country, and probably in others, sporadic or epidemic infections that are not easily recognized by most clinical microbiology laboratories (12). These microorganisms can contribute to build an enteric reservoir of unnoticed transmissible factors of antibiotic resistance that may precede its recognition in routinely searched for pathogens. As an example, in 1970 to 1974, a protracted outbreak of a diarrhea-causing, multiple-drug-resistant S. enterica serovar Typhimurium strain at the "Hospital de Nios" (Montevideo) was preceded by an increasing prevalence of resistance that was observed in EPEC isolates at the same hospital (22).
Thus, the importance of studying these microorganisms from an epidemiological standpoint should be stressed. Efforts must be made to improve the training of technical personnel in antigenic typing techniques of EPEC and to organize regional reference centers capable of performing molecular confirmatory identification.
The antibiotic resistance of these bacteria has to be monitored. The World Health Organization and the Pan American Health Organization have promoted a surveillance program to improve regional knowledge in this field, considering that most major antibiotics may soon be no longer active against bacterial agents responsible for the two main infectious causes of death in children younger than 5 years old in the region: acute respiratory illnesses and diarrhea (15).
Nucleotide sequence accession numbers. The sequences determined in this study were deposited into the EMBL database under accession numbers AJ786366, AJ847362, AJ847363, and AJ847364.
ACKNOWLEDGMENTS
We thank Jorge, Jesús, and Miguel Blanco (LREC, Lugo, Spain) for help in the characterization of EPEC strains. We are grateful to Ana Castro and Elba Hernandez for technical assistance.
This work was partially supported by the Fundacion Manuel Perez, Uruguay, and the Comision Sectorial de Investigacion Científica (CSIC), Universidad de la República, Uruguay. Part of this work was also supported by grants from the LSHM-CT-2003-503335 European project to J.A.A. and a UBACYT, ANPCYT, and Carrillo-Oativia fellowship to G.G., who is a member of the Carrera del Investigador Científico, CONICET-Argentina.
REFERENCES
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
Bauernfeind, A., I. Stemplinger, R. Jungwirth, P. Mangold, S. Amann, E. Akalin, . Ang, C. Bal, and J. M. Casellas. 1996. Characterization of -lactamase gene blaPER-2 which encodes an extended-spectrum class A -lactamase. Antimicrob. Agents Chemother. 40:616-620.
Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513.
Bonnet, R. 2004. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1-14.
Bradford, P. A. 2001. Extended-spectrum -lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933-951.
Decousser, J. W., L. Poirel, and P. Nordmann. 2001. Characterization of a chromosomally encoded extended-spectrum class A -lactamase from Kluyvera cryocrescens. Antimicrob. Agents Chemother. 45:3595-3598.
Gannon, V. P. J., M. Rashed, R. K. King, and E. J. G. Thomas. 1993. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J. Clin. Microbiol. 31:1268-1274.
Gunzburg, S. T., N. G. Tornieporth, and L. W. Riley. 1995. Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene. J. Clin. Microbiol. 33:1375-1377.
Humeniuk, C., G. Arlet, V. Gautier, P. Grimont, R. Labia, and A. Philippon. 2002. -Lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. Antimicrob. Agents Chemother. 46:3045-3049.
Jeong, S. H., I. K. Bae, J. H. Lee, S. G. Sohn, G. H. Kang, G. J. Jeon, Y. H. Kim, B. C. Jeong, and S. H. Lee. 2004. Molecular characterization of extended-spectrum -lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey. J. Clin. Microbiol. 42:2902-2906.
Matthew, M., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of analytical isoelectric focusing for detection and identification of -lactamases. J. Gen. Microbiol. 88:169-178.
Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.
National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, fifth edition. M7-A5, vol. 20. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2000. Performance standards for antimicrobial disk susceptibility tests. Approved standard, seventh edition. M2-A7, vol. 20. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Organizacion Panamericana de la Salud/Organizacion Mundial de la Salud. 1999. Prevention and control of resistance to antimicrobials in America. Report OPS/HCP/HCT/139/99. Organizacion Panamericana de la Salud/Organizacion Mundial de la Salud, Buenos Aires, Argentina.
Paterson, D. L., K. M. Hujer, A. M. Hujer, B. Yeiser, M. D. Bonomo, L. B. Rice, R. A. Bonomo, and the International Klebsiella Study Group. 2003 Extended-spectrum -lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type -lactamases. Antimicrob. Agents Chemother. 47:3554-3560.
Petroni, A., A. Corso, R. Melano, M. L. Cacace, A. M. Bru, A. Rossi, and M. Galas. 2002. Plasmidic extended-spectrum -lactamases in Vibrio cholerae O1 El Tor isolates in Argentina. Antimicrob. Agents Chemother. 46:1462-1468.
Poirel, L., P. Kmpfer, and P. Nordmann. 2002. Chromosome-encoded Ambler class A -lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum -lactamases. Antimicrob. Agents Chemother. 46:4038-4040.
Power, P., M. Radice, C. Barberis, C. de Mier, M. Mollerach, M. Maltagliatti, C. Vay, A. Famiglietti, and G. Gutkind. 1999. Cefotaxime-hydrolysing -lactamases in Morganella morganii. Eur. J. Clin. Microbiol. Infect. Dis. 18:743-747.
Quinteros, M., M. Radice, N. Gardella, M. M. Rodríguez, N. Costa, D. Korbenfeld, E. Couto, G. Gutkind, and the Microbiology Study Group. 2003. Extended-spectrum -lactamases in Enterobacteriaceae in Buenos Aires, Argentina, public hospitals. Antimicrob. Agents Chemother. 47:2864-2869.
Rodríguez, M. M., P. Power, M. Radice, C. Vay, A. Famiglietti, M. Galleni, J. A. Ayala, and G. Gutkind. 2004. Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases. Antimicrob. Agents Chemother. 48:4895-4897.
Schelotto, F., C. M. Rivas, C. Alía, and L. Colensky. 1975. Características de las cepas de S. typhimurium multirresistentes productoras de infeccion cruzada en hospitales pediátricos. Rev. Latinoam. Microbiol. 17:9-16.
Torres, M. E., M. C. Pírez, F. Schelotto, G. Varela, V. Parodi, F. Allende, E. Falconi, L. Dell'Acqua, P. Gaione, M. V. Mendez, A. M. Ferrari, E. Zanetta, H. Chiparelli, and E. Ingold. 2001. Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates. J. Clin. Microbiol. 39:2134-2139.(Rafael Vignoli, Gustavo V)
Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina
Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain
ABSTRACT
We studied 13 extended-spectrum -lactamase (ESBL)-producing enteropathogenic Escherichia coli isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of blaPER-2 and blaTEM-116. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.
TEXT
The emergence of new resistance mechanisms is usually recognized when their corresponding genes reach hospital pathogens, by mobilization or capture of plasmids, transposons, or integrons. These resistance mechanisms are evidenced in the hospital environment only after sudden, and sometimes remarkable, changes in the resistance profile of nosocomial microorganisms. Therefore, it is difficult to define how the corresponding genes gain access to the hospital environment, as resistance is followed primarily in disease-causing microorganisms.
Even if commensally microorganisms play a role in disseminating resistance genes and represent a route for gaining access to pathogenic bacteria, they do not necessarily constitute their ultimate origin. A continuous relationship of those microorganisms with humans may complicate the evaluation of their contribution to resistance, as they may have been submitted to the same antibiotic pressure forces affecting pathogenic bacteria and thus may have gained resistance genes from other microorganisms. Moreover, even environmental microorganisms obtained from most sources may have been in previous contact with bacteria of human origin.
It is accepted that most plasmidic -lactamase-encoding genes (of prevalent broad and extended-spectrum -lactamases and recently carbapenemases) derive from chromosomally located genes of commensal or environmental microorganisms. However, most of these sources are not known, even for the most promiscuous genes, such as blaTEM. Kluyvera species, such as Kluyvera ascorbata, K. cryocrescens, and K. georgiana, have been suggested, for example, as reservoirs of one of the emergent groups of extended-spectrum -lactamases (ESBLs) worldwide: the CTX-M -lactamases (4, 6, 9, 18).
These enzymes appear to be natural ESBLs or "born oxyimino-cephalosporinases" (21), for whose expression there is no need for any mutational shift leading to a modification of the spectrum of activity. The only requisite is to find the right promoter, copy number, or even genetic environment, allowing the proper expression of the -lactamase-encoding gene, and therefore the expected resistance levels for an ESBL. Spread is due to their recruitment or mobilization to more promiscuous elements, such as plasmids harboring transposons or integrons.
Other worldwide prevalent ESBLs, especially TEM- and SHV-derived enzymes, emerge from mutations in genes coding for narrower-spectrum enzymes, already located in transferable elements, resulting in -lactamases with a wider hydrolytic activity (5). Surprisingly, TEM-derived ESBLs have been described only recently in this region (south of Brazil, Argentina, Paraguay, and Uruguay) (16). Additionally, PER-2, the second most prevalent ESBL (20) in isolates from Argentina, has never been reported in any other country.
Acute diarrheal disease is still an important cause of infectious morbidity in Uruguayan children (as well as in many other developing countries), enteropathogenic Escherichia coli (EPEC) being the most frequently found bacterial pathogen. It is usually associated with infants from the poorer social groups; the most prevalent serogroups are O111, O119, and O55 (in decreasing frequency) (23).
Antimicrobial treatment is not recommended for diarrheal diseases caused by enteropathogenic bacteria other than shigellae (i.e., EPEC). Therefore, antibiotic resistance genes harbored by these microorganisms can remain unnoticed and may be silently transferred between different bacterial species in the community or among patients.
We previously reported EPEC isolates resistant to several antibiotics, including 13 oxyimino-cephalosporin-resistant strains, from 68 EPEC strains originally isolated between October 1990 and April 1993. Some characteristics are shown in Table 1. They were obtained from diarrheal cultures of hospitalized children under 30 months old at one pediatric ward of Hospital de Nios "Pereira Rossell" in Montevideo, Uruguay (23).
We focused our attention on the 13 oxyimino-cephalosporin-resistant EPEC isolates, 11 of them belonging to serogroup O119 and the remaining 2 belonging to serogroup O142. Some of their features are displayed in Table 2. All strains were confirmed as eaeA and bfpA positive by PCR (7, 8). MICs, with and without clavulanic acid (4 μg/ml), of ceftriaxone (CRO), ceftazidime (CAZ), and cefepime (FEP) were determined as specified by the NCCLS (13) for isolates showing resistance to CAZ or cefotaxime in disk diffusion tests. Studies of EPEC strain susceptibility to a wide variety of antibiotics were performed by disk diffusion tests following NCCLS guidelines (14).
Antimicrobial susceptibility data suggested a more efficient hydrolysis of CAZ compared to CRO or FEP. Data on gentamicin and trimethoprim-sulfamethoxazole resistance were also compared, rendering three different antibiotypes, as shown in Table 2.
Crude extracts from late-log-phase cultures were obtained from all 13 oxyimino-cephalosporin-resistant EPEC isolates. ESBL characterization was performed by isoelectric focusing (IEF) in broad-range (pH 3 to 10) polyacrylamide gels as previously described (11). All crude extracts displayed only one -lactam-hydrolyzing band (pI 5.4) when revealed with an iodometric overlay system (19) using CRO (1,000 μg/ml) as the substrate.
Guided by IEF results, the presence of blaPER-2 (using primers PER-2F [5'-TGT GTT TTC ACC GCT TCT GCT CTG-3'] and PER-2R [5'-AGC TCA AAC TGA TAA GCC GCT TG-3']) and blaTEM genes (using primers OTR 3 [5'-ATG AGT ATT CAA CAT TTC CG-3'] and OTR 4 [5'-CCA ATG CTT AAT CAG TGA GG-3']) could also be inferred by PCR in each isolate. Plasmid DNA was extracted by alkaline lysis (3). The reaction mixture contained 2 μl of plasmid DNA, and 1.25 U of Taq DNA polymerase (Gibco/BRL) was added to a total volume of 50 μl containing 0.2 mM deoxynucleoside triphosphate, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 50 mM KCl, and 0.5 μM primers. The reaction protocol required an initial step of 5 min at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 20 min. PCR products were automatically sequenced in both strands by primer extension with the same primers, using an ABI PRISM 3700 DNA sequencer. The sequences showed strict identity with blaPER-2 (2) and blaTEM-116 (10).
Plasmid DNA was also used as the template in PCR screening for blaSHV and blaCTX-M using specific primers (1, 19), all results being negative.
PER-2 was first described by Bauernfeind et al. after being sequenced from an Argentinean Salmonella enterica serovar Typhimurium isolate (2). Other microorganisms harboring the same enzyme have been circulating in Buenos Aires since at least 1989; the enzyme was named, at that time, ARG-1 (A. Rossi, M. Quinteros, E. Couto, et al., Abstr. XI Congr. Latinoam. Microbiol., abstr. B49, 1991; M. A. Rossi, G. Gutkind, M. Quinteros, et al., Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 939, 1991). The gene was also recognized in other enterobacteria and related pathogens, including a 1993 Vibrio cholerae isolate (17, 20).
This study represents the first report of PER-2 in a microorganism outside Argentina (however, in a neighboring country, Uruguay).
In addition, we found that TEM-derived ESBLs were already present in Uruguay in the early 1990s. This is the second worldwide TEM-116 clinical report. This enzyme was reported very recently in Korea (10). An interesting coincidence in apparent pIs makes resolution of these two enzymes by IEF analysis very unlikely, perhaps masking one of them just by supposing, from previous data, the existence of the other in the region.
The first blaPER-2-blaTEM-116-carrying-EPEC isolate was obtained in November 1991 during a programmed survey. By the time the epidemiological work was concluded (April 1993) (23), two more groups of isolates carrying the same enzymes but showing different resistance profiles (3 and 1, respectively) were obtained (see details in Table 2). In all three profiles, the initial isolate corresponded to one child stool sample obtained at admission or within 24 h of admission. Other cultures were obtained from hospitalized patients and may correspond to dissemination of these EPEC strains in the ward. However, the three index case cultures are clear indicators of community circulation of the corresponding resistance genes before accessing the hospital. These cultures are probably part of the initial burst of plasmid-borne blaPER-2 diffusion that later involved many bacterial species.
Information about ESBLs in EPEC infections is not readily available. This can be due, at least in part, to the fact that laboratories usually do not devote resources to the identification of EPEC isolates and their antimicrobial susceptibility profile because antibiotic treatment is not indicated for EPEC diarrhea. Thus, data on the resistance of these strains is not routinely produced, as for other bacterial pathogens. Moreover, the attention of microbiologists usually focuses on pathogenic microorganisms but not on related bacteria of indigenous flora that may also develop resistance in treated patients under sustained selective pressure of misguided excessive antibiotic use.
Under these circumstances, EPEC strains are microbial pathogens that can cause in our country, and probably in others, sporadic or epidemic infections that are not easily recognized by most clinical microbiology laboratories (12). These microorganisms can contribute to build an enteric reservoir of unnoticed transmissible factors of antibiotic resistance that may precede its recognition in routinely searched for pathogens. As an example, in 1970 to 1974, a protracted outbreak of a diarrhea-causing, multiple-drug-resistant S. enterica serovar Typhimurium strain at the "Hospital de Nios" (Montevideo) was preceded by an increasing prevalence of resistance that was observed in EPEC isolates at the same hospital (22).
Thus, the importance of studying these microorganisms from an epidemiological standpoint should be stressed. Efforts must be made to improve the training of technical personnel in antigenic typing techniques of EPEC and to organize regional reference centers capable of performing molecular confirmatory identification.
The antibiotic resistance of these bacteria has to be monitored. The World Health Organization and the Pan American Health Organization have promoted a surveillance program to improve regional knowledge in this field, considering that most major antibiotics may soon be no longer active against bacterial agents responsible for the two main infectious causes of death in children younger than 5 years old in the region: acute respiratory illnesses and diarrhea (15).
Nucleotide sequence accession numbers. The sequences determined in this study were deposited into the EMBL database under accession numbers AJ786366, AJ847362, AJ847363, and AJ847364.
ACKNOWLEDGMENTS
We thank Jorge, Jesús, and Miguel Blanco (LREC, Lugo, Spain) for help in the characterization of EPEC strains. We are grateful to Ana Castro and Elba Hernandez for technical assistance.
This work was partially supported by the Fundacion Manuel Perez, Uruguay, and the Comision Sectorial de Investigacion Científica (CSIC), Universidad de la República, Uruguay. Part of this work was also supported by grants from the LSHM-CT-2003-503335 European project to J.A.A. and a UBACYT, ANPCYT, and Carrillo-Oativia fellowship to G.G., who is a member of the Carrera del Investigador Científico, CONICET-Argentina.
REFERENCES
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
Bauernfeind, A., I. Stemplinger, R. Jungwirth, P. Mangold, S. Amann, E. Akalin, . Ang, C. Bal, and J. M. Casellas. 1996. Characterization of -lactamase gene blaPER-2 which encodes an extended-spectrum class A -lactamase. Antimicrob. Agents Chemother. 40:616-620.
Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513.
Bonnet, R. 2004. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1-14.
Bradford, P. A. 2001. Extended-spectrum -lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933-951.
Decousser, J. W., L. Poirel, and P. Nordmann. 2001. Characterization of a chromosomally encoded extended-spectrum class A -lactamase from Kluyvera cryocrescens. Antimicrob. Agents Chemother. 45:3595-3598.
Gannon, V. P. J., M. Rashed, R. K. King, and E. J. G. Thomas. 1993. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J. Clin. Microbiol. 31:1268-1274.
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