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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 1462–1468 0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.5.1462–1468.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.
Plasmidic Extended-Spectrum ␤-Lactamases in Vibrio cholerae O1 Alejandro Petroni,1 Alejandra Corso,1 Roberto Melano,1 Marı´a Luisa Cacace,2 Ana Marı´a Bru,3 Servicio Antimicrobianos, Instituto Nacional de Enfermedades Infecciosas, ANLIS “Dr. Carlos G. Malbrán,” Buenos Aires,1 and Hospital San Vicente de Paul, Orán,2 and Hospital Juan D. Perón, Tartagal,3 Salta, Argentina Received 6 June 2001/Returned for modification 29 September 2001/Accepted 18 January 2002 Since 1992 there have been seven major outbreaks of cholera in Argentina. Susceptibility analysis of 1,947
isolates (40% of reported cases) of Vibrio cholerae O1 biotype El Tor suggested the presence of extended-
spectrum ␤-lactamases (ESBLs) in 28 isolates. Because of their different susceptibility profiles, V. cholerae
isolates M1502, M1516, M1573, and M3030 (all of which are of the Ogawa serotype) were selected for the
present study. By susceptibility analysis, isoelectric focusing, and PCR-based restriction fragment length
polymorphism analysis, CTX-M-type enzymes were identified in three isolates, whereas a PER-2-type enzyme,
in addition to a TEM-1-like enzyme, was identified in the other isolate. The presence of these ESBLs in V.
cholerae isolates resulted in MICs well below those commonly observed for members of the family Enterobac-
teriaceae. Genes that encode both ESBLs were transferred to Escherichia coli by conjugation, together with all
determinants of resistance to non-␤-lactam antibiotics (gentamicin, kanamycin, and sulfamethoxazole for all
isolates; amikacin and streptomycin for three isolates; trimethoprim, tetracycline, and chloramphenicol for two
isolates). Plasmid profile analysis and Southern blotting revealed the presence of single plasmids of about 150
kb in the four V. cholerae isolates and their respective transconjugants and revealed that the plasmids harbored
genes encoding CTX-M-type or PER-2-type ESBLs. These results strongly suggest the broad spread of these
ESBLs among genera belong to families other than the Enterobacteriaceae.
Extended-spectrum ␤-lactamases (ESBLs) have been classi- Pasteran, R. G. Melano, M. F. Galas, M. M. Rodriguez, WHO- fied by Bush et al. (8) in functional group 2be or in Ambler NET-Argentina Collaborative Group, and M. A. Rossi, 39th molecular class A. These enzymes include in their spectra of Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1475, activity penicillins, most cephalosporins, and aztreonam; they 1999). Second, there are the CTX-M ␤-lactamases, which are do not hydrolyze cephamycins (cefoxitin, cefotetan, and moxa- more active against cefotaxime than ceftazidime (for a recent lactam) and carbapenems (imipenem and meropenem); and review, see reference 45). These enzymes have been observed they are inhibited by clavulanic acid, tazobactam, and sulbac- in members of the family Enterobacteriaceae since 1990, and 14 tam (21). Since 1983, plasmidic ESBLs have been extensively members of the family have been reported to date (5, 6, 12, 18, reported worldwide, mainly among species of the family En- terobacteriaceae (17, 21; see http://www.lahey.org/studies/webt Nowadays, CTX-M-producing enterobacteria appear to be .htm). Most plasmidic ESBLs belong to the TEM or SHV type widely spread over very distant geographic regions including (21). Two novel sets of ESBLs that are unrelated to the TEM Japan and Taiwan (45, 49), several European countries (9, 12, and SHV types and that belong to group 2be of Bush et al. (8) 27, 36, 38, 45), South America (5, 6, 45), and very recently, have been observed. First, there are the PER-type enzymes (3, Kenya (18). In particular, CTX-M-2 was broadly detected 25, 26), which are more active against ceftazidime than cefo- mainly in Argentina and also in neighboring countries (31, 35, taxime. PER-1 was isolated from Pseudomonas aeruginosa 45; Galas et al., 38th ICAAC)). Comparisons of the amino acid (26), Salmonella enterica serovar Typhimurium (46), and Aci- sequences of the CTX-M family showed four clusters or netobacter spp. (47), whereas PER-2 was reported in members groups. The first group includes CTX-M-1, CTX-M-3, CTX- of the family Enterobacteriaceae from Argentina (3; M. F. Ga- M-10, and CTX-M-12; the second group comprises CTX-M-2, las, F. G. Pasterán, R. G. Melano, A. E. Petroni, G. Lopez, A. C. Corso, M. A. Rossi, and WHONET Collaborative CTX-M-4 to CTX-M-7, and Toho-1; the third group contains Group, 38th Intersci. Conf. Antimicrob. Agents Chemother., Toho-2, CTX-M-9, and CTX-M-16; and the fourth group con- abstr. E109, 1998; M. F. Galas, M. J. Rapoport, F. G. Pasterán, R. G. Melano, A. E. Petroni, P. G. Ceriana, WHONET-Ar- The broad spread of these enzymes among human patho- gentina Collaborative Group, and M. A. Rossi, 39th Intersci.
genic bacteria which have never been shown to harbor ESBLs Conf. Antimicrob. Agents Chemother., abstr. 1474, 1999; F. G.
could constitute a major health concern and an epidemiologicproblem. Since 1991 Vibrio cholerae O1 biotype El Tor hasbecome one of the most clinically important reemerging patho- * Corresponding author. Mailing address: Servicio Antimicrobianos, gens in Latin America because it has caused cholera outbreaks INEI-ANLIS “Dr. Carlos G. Malbrán,” Av. Velez Sarsfield 563 (1281), and endemic diarrhea. From 1992 to 1998, during seven chol- Buenos Aires, Argentina. Phone and Fax: 54-11-4303-2812. E-mail: era seasons in Argentina, 1,947 isolates of V. cholerae O1 † Dedicated to the memory of Alicia Rossi.
biotype El Tor (40.3% of isolates from reported cases) were EXTENDED-SPECTRUM ␤-LACTAMASES IN V. CHOLERAE TABLE 1. Susceptibility profile distributions for ESC-resistant V. cholerae isolates from the seven Argentinean cholera seasons ESC-resistant isolates (no. of isolates)d a Cholera seasons comprised time periods which generally extended from October to May.
b Reported cases were recorded by the Sistema Nacional de Vigilancia Epidemiológica, Ministerio Nacional de Salud y Acción Social.
c Number of isolates analyzed at the National Reference Laboratory (NRL).
d Resistance or reduced susceptibility are indicated. Abbreviations: AMK, amikacin; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CFP, cefoperazone; CHL, chloramphenicol; CTX, cefotaxime; ERY; erythromycin; GEN, gentamicin; KAN, kanamycin; NIT, nitrofurantoin; O129, vibriostatic agent; SFI, sulfisoxazole; STR, streptomycin; SXT; trimethoprim-sulfamethoxazole; TET, tetracycline.
e ND, no ESC-resistant isolates were detected.
received at the National Reference Laboratory of Argentina.
in 24 cases in seven families. The representative isolate selected for this study Ampicillin resistance was detected in 34 of 1,947 isolates. Sur- (isolate M3030) was isolated from a 48-year-old woman in the first family af- prisingly, 28 of the ampicillin-resistant isolates showed strong fected; she had initially been treated with a single dose of doxycycline.
A susceptible V. cholerae 2717 O1 biotype El Tor isolate used in the suscep- reductions in their susceptibilities to extended-spectrum ceph- tibility profile analysis was isolated (February 1993) from a 16-month-old female alosporins (ESCs) (cefotaxime and/or ceftazidime), cefopera- patient with diarrhea, vomiting, and dehydration symptoms.
zone, and aztreonam. ESC resistance was reversed by clavu- Spontaneous mutants of Escherichia coli C600 and ER1793 (New England lanic acid, suggesting the presence of ESBLs. In this work, we Biolabs, Beverly, Mass.) resistant to nalidixic acid and rifampin were obtained as report on the first identification in V. cholerae of two plasmid- described previously (37) and were used as recipient strains for bacterial conju- mediated ESBLs: CTX-M-type and PER-2-type ESBLs. These Media and chemicals. Mueller-Hinton broth and agar, tryptic soy broth, and
facts strongly suggest the broad spread of these enzymes Luria-Bertani (LB) broth and agar were from Difco (Detroit, Mich.). Restriction among genera belonging to families other than the Enterobac- enzymes were from New England Biolabs. Routine chemicals were from Merck (This study was presented in part at the 38th Interscience Antimicrobial agents and susceptibility testing. The following antimicro-
Conference on Antimicrobial Agents and Chemotherapy, 24 to bial agents were obtained from standard laboratory powders and the indicatedsources: amikacin, aztreonam, and cefepime, Bristol-Myers Squibb; amoxicillin, 27 September 1998, San Diego, Calif. [M. Galas, A. Petroni, R.
Roemmers; clavulanic acid, SmithKline Beecham; ampicillin, Temis Lostaló; Melano, A. Corso, M. Rodriguez, M. L. Cacace, A. M. Bru, chloramphenicol, Parke-Davis; cefotaxime, Argentia; cefoxitin, imipenem, and and A. Rossi, Abstr. 38th Intersci. Conf. Antimicrob. Agents norfloxacin, Merck Sharp & Dohme; ceftazidime, Glaxo; ceftibuten, gentamicin, nitrofurantoin, sulfamethoxazole, and trimethoprim, Schering Plough; cephalo-thin, erythromycin, piperacillin, streptomycin, and tazobactam, Wyeth-Ayerst;kanamycin, Armstrong; sulfisoxazole, Abbott; tetracycline, Microsules y Bern- MATERIALS AND METHODS
abó; and vibriostatic agent O129, Sigma.
MICs were determined by the agar dilution technique according to the spec- Bacterial strains. From February 1992 to June 1998, a total of 1,947 V.
cholerae isolates were received at the National Reference Laboratory (Table 1).
ifications of NCCLS (23) by adoption of the criteria for the family Enterobacte- Susceptibility analysis was carried out by the disk diffusion method according to riaceae for antimicrobial agents other than ampicillin, tetracycline, trimethoprim- the specifications of NCCLS (24) (adopting the criteria for the family Enterobac- sulfamethoxazole, chloramphenicol, and sulfonamides. Clavulanic acid was used teriaceae for antimicrobial agents other than ampicillin, tetracycline, tri- at a ratio of 1 to 2 in combination with amoxicillin or at a constant concentration methoprim-sulfamethoxazole, chloramphenicol, and sulfonamides), and 28 iso- of 4 ␮g/ml in combinations with other ␤-lactams. Tazobactam was used at a lates showed resistance to cefotaxime and/or ceftazidime, cefoperazone, and constant concentration of 4 ␮g/ml.
aztreonam. These V. cholerae isolates (all isolates were of the Ogawa serotype) The following reference strains were used for quality control of dilution tests: were grouped according to their susceptibility profiles, and one isolate from each P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and E. coli group was selected to carry out this study, namely, isolates M1502, M1516, ATCC 25922 and ATCC 35218. Since the MICs of antimicrobial agents for V. M1573, and M3030 (Table 1). V. cholerae M1502 has already been described cholerae 2717 were in the range of those previously reported for susceptible (34). V. cholerae M1516 was isolated from a 43-year-old female outpatient with isolates (34, 48), this isolate was used as a control strain for analysis of the V. vomiting and moderate dehydration in Tartagal, Argentina (north of Salta Prov- cholerae O1 biotype El Tor susceptibility profile.
ince), in March 1993. This isolate was one of three isolates that had identical Conjugation assays. Biparental conjugations were performed as follows. Cells
susceptibility profiles and that had been detected in members of the same family.
of both the donor and the recipient strains were mixed on LB agar at a ratio of V. cholerae M1573 was isolated from a hospitalized newborn male with diarrhea 5 to 1, and the mixture was incubated for 18 h at 35°C. Transconjugants were who had been treated with ampicillin plus gentamicin in Tartagal (April 1993).
selected on Mueller-Hinton agar supplemented with nalidixic acid (50 ␮g/ml) ESC-resistant V. cholerae isolates were not detected during the following three and cefotaxime (8 ␮g/ml) or ceftazidime (0.5 ␮g/ml), as indicated.
cholera seasons (a 4-year period). Toward the end of the sixth cholera season ␤-Lactamase preparation. Bacterial cultures were grown overnight at 35°C in
(March to April 1997) a small outbreak due to an ESC-resistant strain of V. 10 ml of brain heart infusion broth. Cells were harvested by centrifugation, cholerae O1 occurred in Irigoyen and Pichanal, Argentina (north of Salta Prov- washed, and resuspended in 0.5 ml of 10 mM phosphate buffer (pH 7.0). Cell ince). The outbreak began with a 1-year-old male patient who acquired the suspensions were disrupted at 4°C by sonication (two pulses of 45 s at 20 Hz, illness during a hospitalization and rapidly spread into the community, resulting separated by an interval of 30 s, on a Vibra Cell Sonifier [Sonics and Materials Inc., Danbury, Conn.]) and centrifuged (17,000 ϫ g for 10 min at 4°C) for debris buten, and aztreonam were 64, 64, and 16 times higher, re- removal. The supernatants were used for isoelectric focusing (IEF) assays.
spectively, for the group B isolate than for the group A isolates.
IEF of ␤-lactamases. Crude ␤-lactamase extracts were focused on broad-range
In order to analyze if the V. cholerae resistance determinants precast polyacrylamide gels (pH 3.5 to 9.5; Ampholine PAGplate; PharmaciaBiotech, Uppsala, Sweden) with a Multiphor II apparatus (Pharmacia LKB, were harbored in conjugative plasmids, isolates M1502 and Uppsala, Sweden), according to the instructions of the manufacturer. ␤-Lacta- M1573 were mated with E. coli C600 (selections were carried mase bands were visualized by the iodometric method described by Labia and out with nalidixic acid plus cefotaxime for M1502 or with Barthélémy (20), with minor modifications. To differentiate between bands pro- nalidixic acid plus ceftazidime for M1573), rendering transcon- duced by broad-spectrum ␤-lactamase or ESBL activities, after IEF the gel wascovered with an agar overlay containing cephaloridine (1 mg/ml) plus penicillin jugants M1538 and M1561, respectively. In addition, V. chol- (0.1 mg/ml) or ceftazidime (1 mg/ml) plus ceftriaxone (1 mg/ml) as developing erae M1516 and M3030 were conjugated with E. coli ER1793 substrates, respectively. The gel was incubated at room temperature until the (selections were achieved with nalidixic acid plus cefotaxime), bands could be visualized. For ␤-lactamase inhibition procedures, the gel was rendering transconjugants M3099 and M3033, respectively. The covered with a piece of filter paper impregnated with 1 mM clavulanic acid or susceptibility profile of each E. coli transconjugant matched cefoxitin, as indicated, and incubated for 10 min at room temperature beforedevelopment, as described above. The following ␤-lactamases with known pIs that of the respective donor V. cholerae isolate (Table 2). For were used as standards: TEM-1 (pI 5.4), PER-2 (pI 5.4), SHV-2 (pI 7.6), P99 (pI the group A transconjugant strains, the MICs of cefotaxime 7.8), CTX-M-2 (pI 7.9), and SHV-5 (pI 8.2).
(64 to 128 ␮g/ml) and cefepime (16 to 64 ␮g/ml) were the PCR amplifications and PCR-RFLP analysis. Primers specific for the TEM
highest and the MICs of ceftazidime (2 to 8 ␮g/ml), ceftibuten and SHV ␤-lactamase gene families (primers PTEM and PSHV, respectively) (1) were used for PCRs. For amplification of complete structural genes highly (0.5 to 1 ␮g/ml), and aztreonam (16 to 64 ␮g/ml) were the related to that for the CTX-M-2 enzyme (bla lowest. Otherwise, for the group B transconjugant strain, the were designed from its reported DNA sequence (2): 5Ј-CGGAATTCATGATG MICs of ceftazidime (128 ␮g/ml), ceftibuten (32 ␮g/ml), and ACTCAGAGCATTCG-3Ј and 5Ј-GCTCTAGATTATTGCATCAGAAACCG aztreonam (128 ␮g/ml) were the highest and the MICs of TG-3Ј (primers PCTX-M-2 forward and PCTX-M-2 reverse, respectively). For the cefotaxime (8 ␮g/ml) and cefepime (8 ␮g/ml) were the lowest.
identification of the gene encoding PER-2-type enzymes (blaPER-2-type), primers 5Ј-GTAGTATCAGCCCAATCCCC-3Ј and 5Ј-CCAATAAAGGCCGTCCATC For both groups A and B, for either V. cholerae isolates or E. coli transconjugants, clavulanate reduced the MICs nearly PER forward and PPER reverse, respectively) were designed from the reported DNA sequence (3). PCR amplifications were performed with a to the MICs for the susceptible strains.
Perkin-Elmer Cetus thermal cycler in a final volume of 50 ␮l containing 20 pmol Even though selection of transconjugants was achieved only of each primer, 25 ␮M each deoxynucleoside triphosphate, 1.5 mM MgCl2, and 2.5 U of Taq polymerase (Promega, Madison, Wis.). DNA templates were with cefotaxime or ceftazidime, determinants of resistance to prepared by lysing one or two colonies of V. cholerae isolates or E. coli transcon- all non-␤-lactam antibiotics, with the exception of reduced jugants in 50 ␮l of boiling water, as described previously (28, 29). Two microliters susceptibility to nitrofurantoin, shown by V. cholerae M3030, was used for the PCR. The PCR program was 10 min of denaturation at 94°C; were also transferred to E. coli: gentamicin, kanamycin, and 25 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and 30 s of sulfisoxazole for four isolates; amikacin and streptomycin for extension at 72°C; and a final extension step of 5 min at 72°C. The resultingamplification products were run in a 1.5% agarose gel and purified by using a three isolates; and trimethoprim, tetracycline, and chloram- Wizard PCR Preps DNA Purification System (Promega), according to the rec- phenicol for M1516 and M3030, which were also resistant to ommendations of the manufacturer. Purified products were used for PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, as described IEF of ␤-lactamases. IEF of crude homogenates of group A
previously (37), or for construction of the labeled probes used in the hybridiza-tion assays.
V. cholerae isolates (M1502, M1516, and M3030) and their Plasmid profile analysis and hybridization. Plasmid DNA was purified from
respective E. coli transconjugants (M1538, M3099, and M3033) V. cholerae or E. coli transconjugant cells as described previously (22). Plasmidic showed both broad-spectrum ␤-lactamase activities at pI 5.4 DNAs (about 250 ng) were analyzed by electrophoresis in 0.7% agarose gels and ESBL activities at the same pI as that for the CTX-M-2 (Tris-acetate buffer), by using plasmids of known size as standards, and were then standard (pI 7.9). All these activities were inhibited by clavu- transferred onto and immobilized on a nylon transfer membrane (Zeta-ProbeGT; Bio-Rad, Richmond, Calif.). DNA-DNA hybridizations were performed by lanic acid. IEF of crude extracts of V. cholerae M1573 and the method described by Southern (39). Labeling of purified PCR products used E. coli M1561 (group B) revealed the presence of ESBL activ- as probes and stripping and reprobing of the membranes were performed with a ities at the same pI as that for the PER-2 standard (pI 5.4), and Dig DNA Labeling and Detection kit (Boehringer Mannheim, Mannheim, Ger- the activities were also susceptible to clavulanic acid inhibition.
many) by the procedures recommended by the manufacturer.
The presence of a broad-spectrum ␤-lactamase activity at pI 5.4 in M1573 and M1561 could not be investigated by the standard IEF assay because of the overlapping ESBL band at Antibiotic susceptibilities of V. cholerae isolates and conju-
the same pI. Therefore, in situ inhibition with cefoxitin was gational transfer of resistance. The susceptibility profiles of V.
performed on the IEF gel. The ESBL activity at pI 5.4, like the cholerae M1502, M1516, M1573, and M3030 are given in Table activity of the PER-2 standard, was inhibited, whereas the 2. With the exception of the MICs of cefoxitin and imipenem, broad-spectrum ␤-lactamase activity at pI 5.4 and the ESBL the MICs of all ␤-lactams tested were higher for these four isolates than for susceptible isolate 2717. Two different suscep- Identification of ␤-lactamases by PCR-RFLP analysis. Both
tibility profiles for ␤-lactams were recognized, and the four ␤-lactam susceptibility profiles and IEF analysis suggested the isolates were grouped into groups A (V. cholerae M1502, presence of a CTX-M-type enzyme and a PER-like enzyme in M1516, and M3030) and B (isolate M1573). The MICs of the transconjugant strains of groups A and B, respectively.
cephalothin, cefotaxime, and cefepime were eight, four, and Therefore, PCR assays were carried out with total DNA from two times higher, respectively, for the group A isolates than for the four V. cholerae isolates and their respective E. coli the group B isolate, whereas the MICs of ceftazidime and transconjugants as templates and with primers PCTX-M-2, PPER, ceftibuten for the group A isolates were essentially the same as PTEM, or PSHV. When primers PCTX-M-2 were used, only tem- those for V. cholerae 2717. The MICs of ceftazidime, cefti- plates from the group A V. cholerae isolates and their respec- EXTENDED-SPECTRUM ␤-LACTAMASES IN V. CHOLERAE TABLE 2. Antibiotic susceptibilities of V. cholerae isolates and E. coli transconjugants a Abbreviations: AMC, amoxicillin-clavulanic acid; CLA, clavulanic acid; CTB, ceftibuten; CEF, cephalothin; FOX, cefoxitin; FEP, cefepime; IPM, imipenem; NOR, norfloxacin; PIP, piperacillin; TZP, piperacillin-tazobactam; TMP; trimethoprim. The abbreviations for the remaining agents are given in footnote d of Table 1.
b V. cholerae 2717 is a susceptible isolate. E. coli C600 and ER1793 (STRr) were used as recipient strains for bacterial conjugations.
c First-line antibiotics for treatment of V. cholerae infections (4).
d The use of nitrofurantoin is equivalent to the use of furazolidone, the MICs of which for susceptible isolates from Argentina were in the range 0.5 to 0.12 ␮g/ml.
tive transconjugants produced amplification products which To differentiate among these possibilities, we designed a PCR- were of the same size (900 bp), as expected from the compu- RFLP analysis with restriction enzyme BsaHI, which includes tational analysis of the reported DNA sequences of the genes in its recognition sequence the single point mutation between highly related to blaCTX-M-2. The group of highly related genes the CTX-M-2 and the Toho-1 structural genes (Table 3). The comprised genes encoding CTX-M-2 (2), CTX-M-4 (13), amplification products obtained were not digested by BsaHI CTX-M-5 (7), CTX-M-6 and CTX-M-7 (12), and Toho-1 (16).
and, in addition, showed the same restriction map for EcoRV, TABLE 3. PCR-RFLP analysis to differentiate among genes highly related to bla Length(s) (bp) of DNA fragment(s) obtained after digestion with: 500, 310, 90
a The percent identities between blaCTX-M-2 and Toho-1, CTX-M-4, CTX-M-5, CTX-M-6, and CTX-M-7 were 99.9, 98.1, 99.2, 97.5, and 98.4% respectively (NALIGN program; PCGene; IntelliGenetics). PCR products from amplifications with PCTX-M-2 and whole DNA extracted from group A V. cholerae isolates and their derivative transconjugants were incubated with restriction enzymes, and the fragment sizes (boldface type) were estimated after electrophoresis on a 1.8% agarose gel.
The sizes of the restriction fragments from the theoretical PCR products from amplifications with PCTX-M-2 and genes highly related to blaCTX-M-2 were deduced from DISCUSSION
The most common mechanism of resistance to ␤-lactam antibiotics, including recent cephalosporins, is the productionof a ␤-lactamase enzyme. Since 1971 transferable ampicillin resistance have been described in V. cholerae isolates fromAfrica, Asia, and Latin America (10, 19, 32, 33, 42, 43, 44).
However, only two plasmidic ␤-lactamases have been identi- fied to date. First, TEM-1 a broad-spectrum ␤-lactamase was identified in isolates from Asia and Africa (10, 33). Second,SAR-1, a carbenicillinase with ampicillin-hydrolyzing activitylower than that of TEM-1, was described in only two isolatesfrom Tanzania (33). In previous work (34), we reported for thefirst time on the isolation of an ESBL-producing V. choleraestrain (i.e., isolate M1502). In the present work, in addition toa TEM-1-type ␤-lactamase, we have identified two extended- spectrum plasmid-borne enzymes not related to TEM- orSHV-type enzymes in V. cholerae O1 isolates recovered duringseveral cholera seasons in Argentina. For group A isolates, the ␤-lactam susceptibility profiles of the transconjugant strains, FIG. 1. Plasmid profiles and Southern blots of V. cholerae isolates and their derivative transconjugants. Lanes: 1, 3, 5, and 7, V. cholerae for which the cefotaxime/ceftazidime MIC ratio was 32, were isolates M1502, M1516, M1573, and M3030, respectively; 2, 4, 6, and 8, almost identical to those reported for strains producing several E. coli transconjugants M1538, M3099, M1561, and M3033, respec- CTX-M enzymes (16, 45), even though different E. coli recip- tively. (A) Plasmid DNA was extracted as indicated in Materials and ient strains (C600 and ER1793) were used in the mating ex- Methods and analyzed on a 0.7% agarose gel. (B) The gel was trans- ferred and blotted onto a nylon membrane and hybridized against a periments. The results of IEF and PCR-RFLP assays suggest 900-bp PCR product from group A isolates as a probe. (C) The mem- the presence of either CTX-M-2 or a highly related enzyme brane was stripped and reprobed with the 740-bp PCR product from different from those grouped in the CTX-M-2 cluster already the group B isolate. The locations of the loading wells (W) and refer- reported (5, 45). Otherwise, for the group B isolate, the ␤-lac- ence plasmids (numbers indicate sizes [in kilobases]) are shown on the tam susceptibility profile of the transconjugant strain, for which the cefotaxime/ceftazidime MIC ratio was 0.06, was verysimilar to those reported for strains producing PER enzymes HincII, PstI, and SphI compared to that for the bla (26; A. Bauernfeind et al., Abstr. 34th Intersci. Conf. Antimi- crob. Agents Chemother., abstr. C74, 1994). These data, in conjunction with the results of IEF and PCR-RFLP analysis, PER primers generated amplification products (740 bp) only with the templates from the group B V. cholerae isolate are consistent with the presence of a PER-2-type enzyme in V. and its transconjugant. Because of the high degree of DNA cholerae M1561 and its transconjugant, M1573. Cefoxitin inhi- bition of this enzyme, performed on the IEF gel before the assay did not allow differentiation between such genes. A PCR- iodometric method was carried out, allowed the identification RFLP analysis carried out with both group B amplification products and HindIII, PstI, and SphI showed a unique restric- The expression of CTX-M-type and PER-2-type enzymes in tion fragment profile which was identical to that expected for V. cholerae isolates resulted in MICs of ␤-lactams lower than those commonly observed for CTX-M- or PER-2-producing PER-2 gene and different from that expected for the strains that belong to the family Enterobacteriaceae (with the PER-1 gene (data not shown). The use of PTEM primers with all DNA templates produced amplification products of 500 bp, exception of the MICs for ␤-lactams which are poor substrates which is of the expected size of products from a gene encoding for these enzymes, such as cefoxitin and imipenem). This fact was more evident with ESCs, such as cefotaxime, ceftazidime, and cefepime, for which the MICs were 32, 32, and 16 times Plasmid profiles and locations of genes encoding ESBLs.
higher, respectively, for the E. coli transconjugants than for the Analysis of the plasmid profiles of V. cholerae M1502, M1516, V. cholerae isolates. Therefore, differences between V. cholerae M1573, and M3030 and their respective transconjugants and enterobacteria would exist in terms of the levels of expres- (M1538, M3099, M1561, and M3033) showed the presence of sion of ESBLs, the susceptibilities of penicillin-binding pro- single plasmids of about 150 kb in each strain (Fig. 1A). The teins to such ␤-lactam agents, or the periplasmic concentra- DNA hybridization analysis with the 900-bp PCR product from group A isolates as a probe located the gene encoding the Genetic determinants conferring resistance to non-␤-lactam CTX-M-type enzyme on the megaplasmids of group A isolates antimicrobial agents were also encoded by the 150-kb plasmid and their respective transconjugants (Fig. 1B). Reprobing of harbored in each V. cholerae isolate. Multidrug resistance in V. the same membrane with the 740-bp PCR product from the cholerae O1 isolates has so far been linked to plasmids, and the group B isolate as a probe showed that the megaplasmid of this presence of self-transmissible multidrug-resistant plasmids isolate and its transconjugant harbored the gene encoding the from 100 to 200 kb has been reported in V. cholerae O1 isolates from Asia, Africa, and Ecuador (10, 14, 19, 33, 40, 41, 43) and, EXTENDED-SPECTRUM ␤-LACTAMASES IN V. CHOLERAE more recently, in isolates from Albania, Italy, and Brazil (11, showed reduced susceptibility to nitrofurantoin, which was a 15). However, plasmids such those harbored by isolates M1516 common characteristic of isolates from the sixth cholera epi- and M3030, which encode resistance to gentamicin, kanamy- demic in Argentina, but was susceptible to all other antibiotics cin, amikacin, streptomycin, trimethoprim-sulfamethoxazole, tested. Furthermore, an E. coli strain isolated from the urine of tetracycline, and chloramphenicol and, in the case of M1516, the second case patient in the outbreak (the first one from to erythromycin, in addition to ␤-lactam antibiotic resistance, whom an ESC-resistant V. cholerae strain had been isolated) have not previously been reported. Moreover, to the best of showed the same resistance phenotype as that for transconju- our knowledge, gentamicin resistance was reported only in V. gant strain M3033. These data suggest that multidrug-resistant cholerae O1 isolates with two different resistance patterns (R V. cholerae isolates could have resulted from the acquisition of types) from two small outbreaks that occurred in Dacca, Ban- the 150-kb plasmid encoding multidrug resistance in a conju- gladesh, in 1981 (41) and in a single nosocomial pediatric ward gation event with E. coli. The fact that CTX-M-2 is the most in Samutsakorn, Thailand, in 1982 (40). In this work we have frequent ESBL observed in E. coli and members of the family reported on gentamicin resistance in V. cholerae O1 isolates Enterobacteriaceae in Argentina (31, 35; Galas et al., 38th with four different R types from two distantly occurring cholera ICAAC; Galas et al., 39th ICAAC; Pasterán et al., 39th seasons (4 years between the outbreaks). In addition, all the ICAAC; A. C. Gales, T. M. Lewis, J. M. Casellas, V. Prado, J.
ESBL-producing V. cholerae isolates were coresistant to gen- Smayevsky, and R. N. Jones, 39th Intersci. Conf. Antimicrob.
tamicin, as has been observed for ESBL-producing enterobac- Agents Chemother., abstr. 1487, 1999) supports this hypothe- terial isolates from Argentina (Galas et al., 38th ICAAC; Galas sis. In addition, the risks for the potential spread of antimicro- et al., 39th ICAAC; Pasterán et al., 39th ICAAC). This fact bial resistance genes from V. cholerae to other bacteria have suggests that both resistance determinants may be easily co- been pointed out (19). Whatever the case, the findings of this work point out the broad spread of ESBLs among genera The MICs of tetracycline and chloramphenicol for both V. belonging to families other than the Enterobacteriaceae.
cholerae isolates resistant to such drugs (M1516 and M3030) were 32- to 16-fold lower than those for their respective E. coli ACKNOWLEDGMENTS
transconjugant strains (M3099 and M3033, respectively), We thank Norma Binztein and Marta Rivas for providing epidemi- whereas no differences in susceptibilities to aminoglycosides, ologic data from the Argentinean cholera seasons; Marı´a Inés Caffer trimethoprim, and sulfisoxazole were observed between the for serotyping the V. cholerae isolates; and Marisa Rodrı´guez, Gustavo original hosts and the derived transconjugants. This differential López, and Ezequiel Tuduri Franco for technical assistance.
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