RESEARCH LETTER
Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates Gustavo G. Caballero-Flores1, Yaned M. Acosta-Navarrete1, Martha I. Ramı´rez-Dı´az1, Jesu´s Silva-Sa´nchez2 & Carlos Cervantes1 1
Instituto de Investigaciones Quı´mico-Biolo´gicas, Universidad Michoacana, Morelia, Michoaca´n, Me´xico; and 2Centro de Investigacio´n Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pu´blica, Cuernavaca, Morelos, Me´xico
Correspondence: Carlos Cervantes, Instituto de Investigaciones Quı´mico-Biolo´gicas, Universidad Michoacana, Edificio B-3, Ciudad Universitaria, 58030 Morelia, Michoaca´n, Me´xico. Tel./fax: +52 443 326 5788; e-mail:
[email protected] Received 6 September 2011; revised 25 November 2011; accepted 27 November 2011. Final version published online 16 December 2011. DOI: 10.1111/j.1574-6968.2011.02473.x
MICROBIOLOGY LETTERS
Editor: Anthony George Keywords Enterobacteria; plasmid; chromate resistance; chr genes.
Abstract The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in Me´xico. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (CrR), whereas 36% were resistant to mercury (HgR). CrR levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of CrR isolates), encoding a CHR transporter homologue, and merA gene (74% of HgR isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of CrR isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred CrR, as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that CrR genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.
Introduction Resistance to heavy metals is a trait commonly observed in bacteria from diverse environments, including polluted water and soils (Silver & Phung, 2005). In nosocomial bacteria, in addition to the expected genes conferring antibiotic resistance and selected by the use of these agents in therapeutic procedures, genes for resistance to heavy metals may also be present. Thus, bacteria isolated from hospital infections have been found to contain genes that confer resistance to inorganic ions derived from mercury (Porter et al., 1982; Masaru et al., 2004), cadmium (Nucifora et al., 1989), silver (Gupta et al., 2001), and arsenic (Silver et al., 1981), among others. These bacteria possess heavy-metal-resistance genes that are present on chromosomes, plasmids, or transposons (reviewed in Silver & Phung, 2005). Bacterial resistance to hexavalent chromium (chromate; CrO42 ) has been reported mainly in environmental bacteria, including ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
Gram-positive and Gram-negative strains (reviewed in Ramı´rez-Dı´az et al., 2008), although the best studied chromate-resistant mechanism is that encoded by the pUM505 plasmid first identified in Pseudomonas aeruginosa clinical isolates (Cervantes et al., 1990). In this system, a membrane transporter, the ChrA protein, extrudes chromate ions from cytoplasm, thus protecting cells from chromate toxicity (Alvarez et al., 1999). ChrA belongs to the CHR superfamily of transporters, which includes hundreds of members from the three life domains (Dı´azPe´rez et al., 2007); interestingly, ChrA homologues have not been identified in the enterobacterial sequenced genomes. The objective of this study was to evaluate the presence of ChrA homologues in plasmids from a previously characterized collection of antibiotic-resistant enterobacterial isolates of nosocomial origin in an initial attempt to understand the factors that select the prevalence of chromate-resistance genes in bacteria from hospital settings. FEMS Microbiol Lett 327 (2012) 148–154
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Materials and methods
Antibiotic susceptibility of transconjugants was determined as described previously (Miranda et al., 2004).
Bacterial strains and culture conditions
One hundred and nine bacterial isolates causing nosocomial infections were obtained from 14 hospitals in nine major cities in Me´xico during the June 2002 to November 2009 period. These isolates form part of the collection of the Centro de Investigacio´n Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pu´blica (CISEI/ INSP), Cuernavaca, Me´xico (Silva-Sa´nchez et al., 2011) and included the following species (no. of isolates): Escherichia coli (54), Klebsiella pneumoniae (31), Enterobacter cloacae (17), and Salmonella sp. (7). Bacterial clones were assayed by pulse-field gel electrophoresis (PFGE) or by a random amplified polymorphic DNA (RAPD) typing procedure, as described previously (Miranda et al., 2004); the isolates included in this work are representative of the clones defined by these procedures. Clones with identical band patterns were not analyzed in this study. Escherichia coli strain J53-2 (F , met, pro, RifR, CrS) was utilized as metal-sensitive control, as negative control in hybridization assays, and as recipient for plasmid transfer. The P. aeruginosa PAO1 strain (prototroph) bearing plasmid pUM505 (HgR, CrR) (Cervantes et al., 1990) was employed as metal-resistant control and as positive control in colony hybridization assays. Bacterial strains were grown routinely at 37 °C in Luria–Bertani (LB) broth with 1.5% agar added for solid media (Sambrook et al., 1989). Susceptibility tests
For the determination of minimal inhibitory concentrations (MICs), bacterial cultures were grown overnight in LB broth and diluted 1 : 100 in fresh medium, and 25-lL drops was inoculated on LB agar plates with no additions or with increasing concentrations of K2CrO4 (0.2–2 mM) or HgCl2 (25–500 lM) (JT Baker) and incubated for 24 h at 37 °C. MIC was defined as the lowest concentration of the compound that completely inhibited bacterial growth. Chromate susceptibility tests in liquid cultures were performed as follows: overnight cultures grown at 37 °C in nutrient broth (NB; Bioxon, Me´xico) or in LB broth were diluted 1 : 50 in tubes with 4 mL of fresh medium with increasing amounts of K2CrO4 (given their distinct chemical composition, a different range of concentrations of chromate were utilized in each medium). Tubes were incubated at 37 °C for 18 h with shaking, and growth was monitored as optical density at 590 nm with a spectrophotometer. FEMS Microbiol Lett 327 (2012) 148–154
Plasmid procedures
Plasmid DNA from clinical isolates was obtained by alkaline lysis followed by a 60 °C heating step as reported previously (Kieser, 1984) and visualized following electrophoresis in 0.7% agarose gels in TAE buffer (Sambrook et al., 1989). For plasmid transfer by conjugation, log-phase cultures of clinical (donor) isolates and recipient E. coli J53-2 strain were mixed at a 5 : 1 ratio in LB broth and incubated at 37 °C for 24 h without shaking. Transconjugants were selected on LB agar plates with 350 lg mL 1 rifampicin and 2 mM K2CrO4. Plasmid incompatibility groups were determined according to the PCR procedure described by Carattoli et al. (2005). PCR assays were conducted with plasmid DNA isolated from transconjugant strains as described above and a set of oligonucleotide pairs specific for 18 incompatibility groups. Amplification products were visualized following electrophoresis in agarose gels. Inc-group-specific PCR fragments were purified with the Wizard SV and PCR Clean-up System (Promega) and sequenced at the Department of Genetics, CINVESTAV, Irapuato, Me´xico. Hybridization assays
For colony assays, bacteria were inoculated on LB agar plates, and after overnight growth, colonies were lysed with 10% sodium dodecyl sulfate, debris were removed, and DNA was alkali-denatured. DNA was then transferred to nitrocellulose membranes (Hybond-N+; Amersham) and fixed by UV-light exposure. DNA for Southern blot assays was isolated by the alkaline lysis procedure described above, separated by agarose gel electrophoresis, and transferred to nitrocellulose membranes by capillarity. The coding region of the chrA gene was utilized as a probe for chromate-resistance (CrR) genes; a 1.25-kb fragment was PCR-amplified from the pEPL1 plasmid (7.7 kb), which contains a BamHI-PstI 3.8-kb fragment bearing the pUM505 chrA gene cloned in the pUCP20 vector (Ramı´rez-Dı´az et al., 2011). PCR was conducted employing forward oligonucleotide 1D (5′-GAGCGTTG CGAATGAAGAGTCG-3′) and reverse oligonucleotide 1R (5′-GGAAGCATGAAACCGAGTCCC-3′). As a probe for mercury-resistance (HgR) genes, a 1.18-kb fragment comprising most of the merA gene was amplified from pUM505 using forward oligonucleotide MerA-2D (5′-CAT ATCGCCATCATTGGCAGC-3′) and reverse oligonucleotide MerA-2R (5′-CCTCGATGACCAGCTTGATGAAG-3′). PCRs were carried out with Accuprime Super Mix II ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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(Invitrogen) with an initial denaturation for 5 min at 95 °C succeeded by 30 cycles as follows: a denaturation step at 95 °C for 1 min; an annealing step at 60 °C for 45 s, and an elongation step at 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR products were purified as described previously and labeled with the Gene Images AlkPhos Direct Labeling kit (Amersham). Conditions for labeling, hybridization, and signal detection were as recommended by the provider at high stringency (63 °C).
Results and discussion R
To investigate the presence of Cr genes in nosocomial bacteria, a collection of 109 antibiotic-resistant enterobacterial isolates from Mexican hospitals was utilized. This bacterial group was previously characterized by its resistance to multiple antibiotics, including beta-lactams, third-generation cephalosporins, and carbapenems (Miranda et al., 2004; Silva-Sa´nchez et al., 2011). MIC distribution curves demonstrated different levels of chromate susceptibility for each bacterial species (Fig. 1). A clear bimodal distribution of E. coli and K. pneumoniae allowed us to separate CrS from CrR isolates (Fig. 1a and c); for E. cloacae, where a single susceptibility group was found, an arbitrary separation was employed (Fig. 1b). Thus, for E. coli and E. cloacae, species exhibiting a low level of CrR isolates separated from the CrS predominant group, a cutoff value of 1.0 mM chromate was established for isolates to be considered as resistant (Fig. 1a and b); K. pneumoniae isolates displayed a higher number of CrR isolates with a cutoff MIC value at 0.8 mM chro-
No. of isolates inhibited
20 16
8
12
6
8
4
4
2
8
0.2
0.4
0.6
0.8
1.0
1.5 2.0
0 0.0 8
(b)
6
6
4
4
2
2
0 0.0
0.2
No. of isolates with resistance pattern (%) No. hybridizing with probe Bacterial species (no. of isolates)
Cr Hg
E. coli (n = 54)
42 (77.8)
K. pneumoniae (n = 31) E. cloacae (n = 17) Salmonella sp. (n = 7) Total (n = 109)
6 (19.4)
S
S
5 (29.4) 5 (71.4) 58 (53.2)
CrR HgS chrA
CrR HgR chrA+merA
CrS HgR merA
1 1 10 9 1 1 0 – 12 11
1 0 9 8 1 1 0 – 11 9
10 (18.5) 40* 6 (19.4) 6 10 (58.8) 9 2 (28.6) 1 28 (25.7) 20
(1.9) (32.2) (5.9)
(11.0)
(1.9) (29.0) (5.9)
(10.1)
*Five isolates displayed a weak hybridization signal.
mate (Fig. 1c). No isolates in the Salmonella sp. group were considered as CrR because all of them showed a single MIC value of 0.4 mM chromate (Fig. 1d). Using these criteria, 23 isolates (21.1%) were classified as chromate resistant (Table 1). The MIC distribution curve for mercury showed a clear bimodal susceptibility pattern: a group of HgS isolates with MICs of 25–50 lM HgCl2 and a group of HgR isolates with MICs at 300–400 lM HgCl2 (Supporting information, Fig. S1). The HgR group consisted of 39 isolates (35.8%; Table 1). MIC analysis showed that nearly one-half of the isolates (51/109) were resistant to either chromate or mercury, whereas 11 isolates (10.1%) displayed resistance to
10 (c)
(a)
0 0.0
Table 1. Chromate- and mercury-resistance patterns and hybridization with chrA and merA probes of the clinical isolates
0.4
0.6
0.8
1.0
1.5 2.0
0.2
0.6
0.8
1.0
1.5 2.0
(d)
0 0.0
0.2
MIC (mM chromate)
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0.4
0.4
0.6
0.8
1.0
1.5 2.0
Fig. 1. Distribution of the MIC of chromate in nosocomial isolates. Isolates were grown as described in the Materials and methods, and the MIC of K2CrO4 was determined after a 24-h incubation. (a) Escherichia coli; (b) Enterobacter cloacae; (c) Klebsiella pneumoniae; (d) Salmonella sp. Isolate numbers for each species are provided in Table 1. Each value represents the average of three independent assays. Vertical dotted lines indicate the MIC cutoff points that distinguish sensitive from resistant isolates.
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both agents (Table 1). The proportion of HgR isolates was similar to that found in other collections of nosocomial bacteria (ranging from 30% to 50%; Porter et al., 1982; Deredjian et al., 2011). Possible sources of chromate acting as selective factors in hospital settings are not recognizable. The mechanisms of selection of heavymetal-resistance phenotypes within hospitals remain unclear (Porter et al., 1982; Yurieva et al., 1997), but the nosocomial use of metal derivatives (i.e. organomercurials, silver compounds) as antiseptics or disinfectants has been proposed as a selective factor. The majority of E. coli and Salmonella isolates were metal sensitive, whereas metal-resistant isolates predominated in K. pneumoniae (61.3% CrR; 48.4% HgR) (Table 1). Klebsiella pneumoniae isolates have been considered as reservoirs of antibiotic-resistance plasmids in hospital settings (Carattoli, 2009), which may be related to the high levels of resistance to metals observed in this species. Enterobacter cloacae isolates had a different pattern: the majority were chromate sensitive (11.8% CrR), but exhibited the highest proportion (64.7%) of mercury resistance (Table 1). The presence of CrR genes in the nosocomial isolates was first screened by colony hybridization assays at high stringency, utilizing a probe designed from the chrA gene from P. aeruginosa pUM505 plasmid (Ramı´rez-Dı´az et al., 2011). Widespread distribution of pUM505-related chrA genes in a previous study with P. aeruginosa clinical isolates from Me´xico (Cervantes & Ohtake, 1988) suggested that utilization of such a probe might allow for the detection of chrA sequences in the current collection under the conditions employed. Hybridization signals were found in
20/23 (86.9%) of the CrR isolates (data not shown and Table 1), suggesting that the majority possessed a mechanism of resistance involving chromate efflux. chrA sequences were detected in isolates from three of the four bacterial species analyzed (Table 1), indicating wide distribution of chrA homologues in the enterobacterial collection. A similar assay with a probe derived from the merA gene from pUM505 showed that nearly 75% of HgR isolates (29/39) hybridized; five E. coli HgR isolates showed weak hybridization signals, suggesting that they may contain merA homologues with lower similarity to the probe (data not shown and Table 1). These data suggest that the majority of HgR isolates possess a mechanism of resistance involving inorganic-mercury reduction. It has been proposed that linkage of metal-resistance genes with antibiotic-resistance genes in mobile genetic elements, such as plasmids and transposons, may allow for coselection owing to antimicrobial use (Baker-Austin et al., 2006). Because CrR genes usually reside on plasmids, CrR isolates that hybridized with the chrA probe (hereafter denominated chrA+ isolates) were analyzed for plasmid content. Of the 20 chrA+ isolates, nine showed from one to five plasmid bands each, ranging in size from five to 100 kb (some examples are shown in Fig. 2a). The remaining 11 isolates that did not yield plasmid bands by the DNA extraction procedure employed were not further studied. Southern blot assays utilizing the same probe and conditions as in colony hybridizations were then carried out with the nine chrA+ isolates exhibiting plasmid bands. The pEPL1 (chrA+) plasmid showed several bands in the agarose gel and the Southern blot, which
(a)
M1 M2 K78
K86 En94 K99 K120 +
(b)
–
K78 K86 En94 K99 K120 +
–
93 45 CF
7.7
Fig. 2. Plasmid profiles and Southern blot hybridization of chrA+ isolates. (a) Total DNA was extracted as described under Materials and methods and separated by agarose gel electrophoresis. Names above lanes indicate species abbreviation (K, Klebsiella pneumoniae; En, Enterobacter cloacae) and isolate name as shown in Table 2. Controls: (+), pEPL1 (chrA+) plasmid; ( ), Escherichia coli J53-2. The gel was loaded with 100– 300 ng of DNA from nosocomial isolates, 500 ng of DNA from E. coli J53-2, and 5 ng of pELP1 plasmid DNA. Positions of plasmids 2F10 (45 kb; lane M1) and R1 (93 kb; M2), used as molecular size markers, and pEPL1 (7.7 kb), are indicated to the left. Location of chromosomal fragments (CF) is also noted. (b) Southern blot hybridization of the gel in (a) was carried out at 63 °C with the chrA probe, as described in the Materials and methods. Notice that control samples were hybridized separately to avoid overexposure of the positive control.
FEMS Microbiol Lett 327 (2012) 148–154
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(a) 1.0
OD590 nm
0.8 0.6 0.4 0.2 0.0
0.0
0.1
0.2
0.3
0.4
0.5
Chromate (mM) (b) 1.2 1.0
OD590 nm
0.8 0.6 0.4 0.2 0.0
0.0
0.2
0.4
0.6
0.8
1.0
Chromate (mM) Fig. 3. Chromate susceptibility of Escherichia coli transconjugants with plasmids from chrA+ isolates. Cultures were grown in nutrient broth (a) or LB broth (b) with the chromate concentrations indicated at 37 °C for 18 h with shaking, and the optical density (OD) at 590 nm was determined. (a) E. coli J53-2 (●); transconjugant of J532 with 100-kb plasmid from Enterobacter cloacae 94 (Δ); transconjugant of J53-2 with 80-kb plasmid from Klebsiella pneumoniae 78 (□). (b) E. coli J53-2 (●); transconjugant of J53-2 with 85-kb plasmid from K. pneumoniae 99 (Δ); transconjugant of J53-2 with 95-kb plasmid from K. pneumoniae 86 (□). Each value represents the average of two independent assays performed in duplicate with standard error (SE) bars shown.
corresponded to distinct topologic plasmid forms (Fig. 2, + lanes). Five of the isolates displayed hybridization signals in both plasmid bands (from 40 to 100 kb) and chromosomal DNA fragments (Fig. 2b).
Although both plasmid and chromosomal chrA homologues have been identified in diverse bacteria (Ramı´rezDı´az et al., 2008), we next focused only on plasmidic chrA genes from chrA+ isolates. Single plasmids from three K. pneumoniae isolates and from one E. cloacae isolate, with a common geographic origin but of different isolation date and molecular size (Table 2), were transferred by conjugation to the E. coli J53-2 RifR strain selecting for CrR. Plasmids of 40 and 90 kb from isolate K. pneumoniae 120, which hybridized with the chrA probe (Fig. 2b, lane K120), could not be transferred to J53-2 and were not further analyzed. Besides CrR, the four plasmids that could be transferred also conferred resistance to multiple antibiotics (Table 2), all of them already known to be present in the parental clinical isolates (Miranda et al., 2004; Silva-Sa´nchez et al., 2011). Escherichia coli transconjugants obtained from the four chrA+ isolates showed single plasmid bands in agarose gels (Fig. S2) and a CrR phenotype in chromate susceptibility tests. Figure 3a depicts the results obtained with transconjugants from K. pneumoniae 78 and E. cloacae 94 isolates, which tolerated higher chromate levels when grown in NB medium, as compared with the E. coli J53-2 plasmidless strain; under the same growth conditions, transconjugants from K. pneumoniae 99 and 86 behaved as chromate sensitive (data not shown), although displayed moderate but reproducible chromate resistance when tested in LB broth (Fig. 3b). Differential expression of chrA homologues from host cells grown in different culture media has been reported previously (Aguilar-Barajas et al., 2008); a possible role of sulfate levels on differential expression has been postulated. To our knowledge, this is the first report of plasmids from enterobacteria bearing functional chrA genes. chrA genes are widely distributed among organisms, ranging from bacteria to archaea and to fungi (Dı´az-Pe´rez et al., 2007). In the case of bacteria, chrA genes are broadly allocated in species of proteobacteria, cyanobacteria, actinobacteria, and firmicutes (Dı´az-Pe´rez et al., 2007; Henne et al., 2009); however, although chrA homologues have been identified in enterobacteria, they are only present in plasmids (Nies et al., 2006). From 69 enterobacterial genomes sequenced to date (NCBI database), only one (from K. pneumoniae KCTC 2242) possesses a
Table 2. Properties of conjugative plasmids from chrA+ isolates Isolate name K. pneumoniae 78 K. pneumoniae 86 E. cloacae 94 K. pneumoniae 99
Origin* Monterrey Monterrey Monterrey Monterrey
Plasmid size (kb) (2008) (2006) (2007) (2007)
80 95 100 85
Resistance pattern Cr, Cr, Cr, Cr,
Ap, Ap, Ap, Ap,
Ctx, Ctx, Ctx, Ctx,
†
Km , CAZ, Tc Km, Gm Km, CAZ, Tc, CIP Km, Gm
Inc group IncN IncP IncN/P IncP
Cr, chromate; Ap, ampicillin; Ctx, cefotaxime; Km, kanamycin; CAZ, ceftazidime; Tc, tetracycline; Gm, gentamicin; CIP, ciprofloxacin. *City of origin (year of isolation). † A common resistance pattern is shown underlined.
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chromosomal chrA homologue; nine additional chrA homologues reported in the database were identified in plasmids from five different enterobacterial species. We have no explanation for this phenomenon yet, but it appears that an enterobacterial ancestral genome may have lost chrA genes, probably by the lack of selective pressure because of chromate exposure; under this situation, enterobacterial strains might possess chrA genes solely when carried on mobile elements. Transferable CrR plasmids were classified according to their incompatibility groups by a PCR-based procedure. The appearance of specific amplification products demonstrated that they belonged to the groups IncN (80-kb plasmid from K. pneumoniae 78) and IncP (95- and 85-kb plasmids from K. pneumoniae 86 and 99) (Fig. S3). The 100-kb plasmid from E. cloacae 94 displayed amplification fragments from both IncN and IncP groups and was classified as a hybrid IncN/P plasmid. IncP and IncN/P plasmids yielded a second unspecific PCR product, but DNA sequencing confirmed the identity of the 534-pb fragment with IncP-group replicons (Fig. S3). The p80 IncN plasmid showed an antibiotic-resistance pattern similar to that of the IncN/P plasmid, except that the latter conferred additional ciprofloxacin resistance (Table 2); these data suggest that the IncN/P plasmid may have resulted from recombination between IncN and IncP K. pneumoniae plasmids. IncP plasmids have been reported to participate in recombination events with other replicons (Schluter et al., 2003). The two IncP plasmids shared a similar antibiotic-resistance pattern (Table 2), which also suggests a genetic relatedness between them. IncN plasmids are considered of intermediate host range and are frequently found only in Enterobacteriales, whereas IncP plasmids have a rather broad host range (Suzuki et al., 2010). The chrA gene from pUM505 plasmid, in addition to being located on a conjugative replicon, forms part of a putative transposon (Ramı´rez-Dı´az et al., 2011). Moreover, besides CrR, pUM505 has been shown to encode HgR, oxidative stress protection, and virulence properties (Ramı´rez-Dı´az et al., 2011), which may represent additional adaptive traits that promote distribution of the plasmid, or its genes, among nosocomial bacteria. chrA gene homologues from plasmids of Pseudomonas sp. (Tauch et al., 2003) and Comamonas sp. (Ma et al., 2007), as well as from the chromosomes of Ochrobactrum tritici 5bvl1 (Branco et al., 2008), Bacillus cereus SJ1 (He et al., 2010), and Pseudomonas sp. (Petrova et al., 2011), are also located on putative transposable elements. In conclusion, our results showed that chrA gene homologues are frequently found in plasmids of enterobacterial isolates of nosocomial origin and suggest that CrR genes may be transferred among hospital bacteria FEMS Microbiol Lett 327 (2012) 148–154
owing to their location within genetic mobile elements, probably coselected by antibiotic exposure.
Acknowledgements The present work was partially supported by grants from Coordinacio´n de Investigacio´n Cientı´fica (UMSNH; 2.6 and 2.35) and Consejo Nacional de Ciencia y Tecnologı´a, Me´xico (Conacyt no. 79190). GGC-F and YMA-N were recipients of postgraduate and graduate fellowships from Conacyt, respectively.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Distribution of the Minimal inhibitory concentration (MIC) of mercury in nosocomial isolates. Figure S2. Transfer of plasmids by conjugation. Figure S3. Determination of incompatibility groups of plasmids from chrA+ transconjugants. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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