Characterization of mercury-resistant clinical Aeromonas species

SHORT COMMUNICATION

Characterization of mercury-resistant clinical Aeromonas species

Abigail Pérez-Valdespino; Martin Celestino-Mancera; Viridiana Lorena Villegas-Rodríguez; Everardo Curiel-Quesada

Department of Biochemistry, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico

^{Correspondence}

ABSTRACT

Mercury-resistant Aeromonas strains isolated from diarrhea were studied. Resistance occurs via mercuric ion reduction but merA and merR genes were only detected in some strains using PCR and dot hybridization. Results indicate a high variability in mer operons in Aeromonas. To our knowledge, this is the first report of mercury-resistant clinical Aeromonas strains.

Key words:Aeromonas, mercury resistance, clinical strains.

Mercury is one of the most toxic of the heavy metals. Mercury toxicity arises from the avid ligation of its compounds to the thiol groups in proteins, which results in inhibiting macromolecule synthesis and enzyme action. Bacteria have developed several mechanisms to neutralize mercury toxicity, the most common being the conversion of highly toxic mercuric ions to less toxic and volatile elemental mercury through chemical reduction. Mercuric ion resistance involves a diverse set of genes, which are widespread in both Gram-positive and Gram-negative bacteria. The bacterial mer operons encode a cluster of genes involved in the detection, mobilization and enzymatic detoxification of mercury (Barkay and Wagner-Döbler, 2005).

Bacteria belonging to the genus Aeromonas are Gram-negative metabolically versatile bacilli, able to adapt and colonize different terrestrial and aquatic ecosystems. In accordance with this ubiquity, Aeromonas species resistant to a great variety of antibiotics and heavy metals have been described. Some species of this genus are considered pathogens responsible for various infections in humans (Janda and Abbott, 2010). This work focused on the isolation and characterization of mercury resistant Aeromonas from human diarrheic feces where no other known enteropathogenic bacterium was isolated.

Forty-one clinical strains were isolated from diarrheic feces collected between July and October 2005 from patients attending the Public Health Institute at Hidalgo State, Mexico. Strains were classified as Aeromonas by standard bacteriological tests. Mercury resistant strains (n = 28) were further identified using the 16S rDNA-RFLP pattern (Figueras et al., 2009). Species assignation were completed by PCR amplification and sequencing of rpoD gene. Strains not yielding an rpoD amplicon were identified by gyrA amplification and sequencing (Beaz-Hidalgo et al., 2010). Using these approaches, the following species were found: A hydrophila (n = 11), A. caviae (n = 10), A. veronii (n = 3), A. aquariorum (n = 3) and A. media (n = 1).

The susceptibility profile to five different antimicrobial agents was tested by the Kirby-Bauer disk diffusion method (Table 1) (CLSI 2007). Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 25923, Staphylococcus aureus ATCC 27853 and A. hydrophila ATCC 7966 were used as reference strains.

In order to determine the minimum inhibitory concentration of mercury (MIC), cultures were grown overnight in trypticase soy broth (TSB) and then 10 μL of each culture was spotted onto brain heart infusion (BHI) (Oxoid Hampshire, England) agar plates containing 0 to 600 mg mL^-1 HgCl₂. Plates were incubated at 37 °C. The lowest concentration that inhibited growth was considered the MIC. Results are shown in Table 1. Since all strains (N = 41) were able to grow in the presence of 10 μg mL^-1 HgCl_2, the cutoff value for mercury resistance was set at 25 μg mL^-1.

All mercury-resistant Aeromonas strains were tested in a mercury volatilization assay by the X-ray film method as described (Nakamura and Nakahara, 1988). All 28 mercury-resistant strains were able to reduce mercuric ions as shown by the appearance of dark spots on the X-ray film, resulting from the formation of a silver amalgam (Table 1).

The presence of merA and merR genes was assessed by PCR amplification with primers previously described (MerA1, MerA2, A1s-n.F and A5-n.R) (McIntosh et al., 2008; Vetriani et al., 2005) or designed in this work (MerA.1F: CGACTGCACCGACCAGCC, MerA.1R: AAGGTCTGCGCCGCGAGCT, MerR.1F: ATCCGBTTCTATCAGCGCAAG and MerR.1R: ACGTCCTTNRGCTTGTGCTCG). Isolation of total DNA from metal-resistant strains was done according to standard protocols (Sambrook and Russell, 2001). PCR reactions were done with PCR Master Mix (2X) (Fermentas, Lithuania) and amplifications involved standard cycles of PCR.

Using MerA.1F and MerA.1R primers 12 out of 28 strains yielded the expected 461 bp amplicon. Strains that did not yield an amplification product with these primers were subjected to a new amplification reaction with primers MerA1 and MerA2. Using these primers, amplicons with the expected size were obtained for three additional strains. In an further attempt to amplify the merA gene from total DNA of thirteen negative strains, degenerate primers A1s-n.F y A5-n.R were used in new merA amplification reactions. Five of the strains tested this way rendered an amplification product of the right size. However, sequencing of these fragments followed by alignment to the Gen Bank database revealed no similarity with any gene belonging to the mer operon. The merR gene amplification assays yielded the right amplicon in 17 mercury-resistant strains. Not all the strains positive for the merA gene rendered a product for the merR gene or viceversa (Table 1).

The presence of genes coding for MerA and MerR was also explored using a dot hybridization assay. DNA probes to detect merA and merR homologues were synthesized by PCR amplification of A. salmonicida AS03 total DNA with MerA1 and MerA2 or MerR.1F and MerR.1R primers, respectively, in the presence of digoxigenin-dUTP. Dot blot hybridization was conducted at high stringency according to the instructions of the manufacturer (Digoxigenin-High Prime DNA Labeling and Detection Starter Kit I; Roche Molecular Biochemicals, Indianapolis, IN). All strains produced positive hybridization signals for merA but none for merR gene (Table 1).

In order to know whether mercury resistance was carried by an extrachromosomal element, plasmids were extracted from all the mercury-resistant strains according to Birnboim and Doly (Birnboim and Doly, 1979) and Kado and Liu procedures (Kado, and Liu, 1981). Eight plasmid-bearing strains with sizes ranging from 3 to 53 kb were found. These strains were used as donors in conjugal transfer experiments. Matings were performed on sterile nitrocellulose filters on TSA plates using E. coli J53-1 Nal^R as a recipient. Transconjugants were selected on TSA plates containing 30 μg mL^-1 nalidixic acid and 50 μg mL^-1 HgCl₂. A. salmonicida AS03 bearing a conjugative plasmid conferring mercury resistance was used as a positive control. In this case matings were performed at 28 °C. No mercury-resistant transconjugants could be detected in any of the mating experiments. In view of these results we intended to introduce the plasmids by bacterial transformation. Transformation experiments were as described by Chung et al. (1993), using E. coli DH5α (Invitrogen CA, USA) as host for the incoming plasmids. Transformants were selected on TSA plates supplemented with 50 μg mL^-1 HgCl₂. Transformation using plasmid preparations did not yield any mercury-resistant E. coli colonies. In agreement with these results, attempts to PCR amplify merA or merR genes from plasmid bands excised from agarose gels were unsuccessful. These results show that plasmids are not related with metal resistance.

Multiple reports on bacterial resistance to mercury including diverse bacterial groups exist in scientific literature. However, works on mercury resistance in Aeromonas are scarce (De J Ramaiah et al., 2003; Huddleston et al., 2006; McIntosh et al., 2008). In this work mercury-resistant strains were characterized as: A. hydrophila, A. caviae, A. veronii, A. aquariorum and A. media. The first three species have been reported as the most frequently associated to diarrhea in Mexico (Aguilera-Arreola et al., 2007). Some species of Aeromonas present high intraspecific diversity and the presence of different ecotypes has been suggested depending on the strain provenance. Therefore our strains were probably able to acquire the genes for adaptation to mercury-rich environments from diverse sources (Aguilera-Arreola et al., 2005).

Different works on the isolation of bacteria resistant to heavy metals as part of normal human biota or clinical specimens exist. These works were performed using a variety of culture media (Deredjian et al., 2011; Pike et al., 2002). Since it has been reported that results on resistance levels are highly affected by the culture medium used (Pike et al., 2002a); a direct comparison of mercury resistance among strains is not easy. In our experimental conditions strains resistance ranged between 25 and 600 μg mL^-1. HgCl₂. Bacteria growing in the presence of 25 μg mL^-1 are true resistants as confirmed by the mercury volatilization assay and by the amplification of the merA or merR genes. Some of these bacteria are particularly resistant comparing to strains described recently by McIntosh et al. (2008). They isolated several highly resistant A. salmonicida strains able to grow in 270 μg mL^-1 HgCl₂. Using the same medium and one of these strains (AS03), as a positive control, we found several clinical isolates able to grow between 300 to 600 μg mL^-1 HgCl_2. Similar levels of resistance as observed in our aeromonad isolates have only been reported in Bacillus subtilis strains, which are able to grow in medium containing up to 550 μg mL^-1 HgCl₂ (Kannan and Krishnamoorthy, 2006).

Many papers evidence the frequent association between the mer operon and antibiotic resistance determinants (Baker-Austin et al., 2006; Mathema et al., 2011). Given that several mercury-resistant clinical strains isolated in this work showed also resistance to tetracycline, trimethoprim, nalidixic acid and streptomycin (Table 1), a search for plasmids was implemented. We found plasmids in some mercury-resistant strains. Mercury resistance could not be transferred by conjugation or transformation with plasmid DNA, which suggests that no relationship between these mobile elements and the metal-resistance phenotype exists. This was confirmed by the lack of amplification of merA or merR genes using plasmid DNA as a template. Our results contrast with reports in literature where mer operons reside frequently in plasmids (Nakahara et al., 1977). Previous work in our laboratory searching for Class 1 integrons revealed also a preferential chromosomal vs. plasmid localization of these elements in Aeromonas spp. strains (Pérez-Valdespino et al., 2009).

Mercury reduction is the most common mercury detoxification mechanism in bacteria. All strains were able to reduce the mercuric ion to elementary mercury. Our results show a widespread distribution of mercury reducing activity in the clinical Aeromonas spp. strains studied. Despite these results, we could not amplify the merA gene in around half of the strains using three different primer sets, including primers A1sn.F and A5n.R, which are widely used to demonstrate the presence of the merA gene in an large diversity of bacteria (Ní Chadhain et al., 2006; Skurnik et al., 2010; Vetriani et al., 2005). Using these primers we found amplicons of the right size derived from some of our clinical Aeromonas strains. However, these amplicons showed sequences not related to merA. We suggest that amplicons generated with these primers should always be sequenced in order to avoid false positive results. This is in accordance with Rochelle et al. (Rochelle et al., 1991), who recommend confirming the presence of the mer genes using several techniques or probes. Due to the failure to amplify the merA gene in several strains, we searched for the presence of the merR gene. As observed with merA, not all strains yielded the merR amplicons. Considering these results, we resorted to search for merA and merR homologues through dot hybridization assays. All strains hybridized with the merA probe but none of them hybridized with the merR probe. Taken together, these results suggest a high variability of mer operons in clinical strains. Mercury exerts a selective pressure on microorganisms, which facilitates the selection of resistant strains carrying genetic elements conferring resistant to mercury from different bacteria to Aeromonas, accounting for this variability (Baker-Austin et al., 2006; Smit et al., 1998). Highly resistant microorganisms play a key role in mercury detoxification and mobilization in natural environments. Isolation of highly mercury-resistant Aeromonas from diarrheic feces might result from human exposure to water heavily contaminated with the metal, which would pose a threat to human health.

Acknowledgments

We are grateful to Dr. Frank Fekete, Department of Biology, Colby College Waterville, ME for the generous gift of A. salmonicida AS03. This work was supported by ICyTDF 254/2009 and SIP 20110799. A. Perez-Valdespino is a PhD CONACYT scholarship recipient. E. Curiel-Quesada is a COFAA and EDI fellow. Competing interests: None declared. Ethical approval: Not required.

Submitted: July 4, 2012

Approved: April 4, 2013

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License CC BY-NC.

• Aguilera-Arreola MG, Hernández-Rodríguez C, Zúñiga G, Figueras MJ, Castro-Escarpulli G (2005) Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes. FEMS Microbiol Lett 242:231-240.
• Aguilera-Arreola MG, Hernández-Rodríguez C, Zúñiga G, Figueras MJ, Garduño RA, Castro-Escarpulli G (2007) Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical strains from Mexico and Spain: a comparative study. Can J Microbiol 53:877-887.
• Beaz-Hidalgo R, Alperi A, Buján N, Romalde JL, Figueras MJ (2010) Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish. Syst Appl Microbiol 33:149-153.
• Barkay T, Wagner-Döbler I (2005) Microbial transformations of mercury: potentials, challenges, and achievements in controlling mercury toxicity in the environment. Adv Appl Microbiol 57:1-52.
• Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513 - 1523.
• Baker-Austin C, Wright MS, Stepanauskas R, Mc Arthur JB (2006) Co-selection of antibiotic and metal resistance. Trends Microbiol 14:176-182.
• Clinical Laboratory Standard Institute (2007) Performance standards for antimicrobial susceptibility testing. Wayne, PA, USA.
• Chung CT, Miller RH (1993) Preparation and storage of competent Escherichia coli cells. Methods Enzymol 218:621-627.
• De J Ramaiah N, Mesquita A, Verlekar XN (2003) Tolerance to various toxicants by marine bacteria highly resistant to mercury. Mar Biotechnol 5:185-193.
• Deredjian A, Colinon C, Brothier E, Favre-Bonté S, Cournoyer B, Nazaret S (2011) Antibiotic and metal resistance among hospital and outdoor of Pseudomonas aeruginosa Res Microbiol 162:689-700.
• Figueras MJ, Alperi A, Saavedra MJ, Ko WC, Gonzalo N, Navarro M, Martínez-Murcia AJ (2009) Clinical relevance of the recently described species Aeromonas aquariorum J Clin Microbiol 47:3742-3746.
• Huddleston JR, Zak JC, Jeter RM (2006) Antimicrobial susceptibilities of Aeromonas spp. isolated from environmental sources. Appl Environ Microbiol 72:7036-7042.
• Janda JM, Abbott SL (2010) The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 23:5-73.
• Kannan SK, Krishnamoorthy R (2006) Isolation of mercury resistant bacteria and influence of abiotic factors on bioavailability of mercury a case study in Pulicat Lake North of Chennai, South East India. Sci Total Environ 367:341-353.
• Kado CI, Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365-1773.
• Mathema VB, Thakuri BC, Sillanpä M (2011) Bacterial mer operon-mediated detoxification of mercurial compounds: a short review. Arch Microbiol 193:837-844.
• McIntosh D, Cunningham M, Ji B, Fekete FA, Parry EM, Clark SE, Zalinger ZB, Gilg IC, Danner GR, Johnson KA, Beattie M, Ritchie R (2008) Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254. J Antimicrob Chemother 61:1221-1228.
• Nakahara H, Ishikawa T, Sarai Y, Kondo I, Kozukue H (1977) Mercury resistance and R plasmids in Escherichia coli isolated from clinical lesions in Japan. Antimicrob Agents Chemother 11:999-1003.
• Nakamura K, Nakahara H (1988) Simplified X-ray film method for detection of bacterial volatilization of mercury chloride by Escherichia coli Appl Environ Microbiol 54:2871-2873.
• Ní Chadhain SM, Schaefer JK, Crane S, Zylstra GJ Barkay T (2006) Analysis of mercuric reductase (merA) gene diversity in an anaerobic mercury-contaminated sediment enrichment. Environ Microbiol 8:1746-1752.
• Pérez-Valdespino A, Fernández-Rendón E, Curiel-Quesada E (2009) Detection and characterization of class 1 integrons in Aeromonas spp. isolated from human diarrheic stool in Mexico. J Basic Microbiol 49:572-578.
• Pike R, Lucas V, Stapleton P, Gilthorpe MS, Roberts G, Rowbury R, Richards H, Mullany P, Wilson M (2002) Prevalence and antibiotic resistance profile of mercury-resistant oral bacteria from children with and without mercury amalgam fillings. J Antimicrob Chemother 49: 777-783.
• Pike R, Stapleton P, Lucas V, Roberts G, Rowbury R, Richards H, Mullany P, Wilson M (2002a) Effect of medium composition on the susceptibility of oral streptococci to mercuric chloride. Curr Microbiol 45:272-276.
• Rochelle PA, Wetherbee MK, Olson BH (1991) Distribution of DNA Sequences Encoding Narrow- and Broad-Spectrum Mercury Resistance. Appl Environ Microbiol 57:1581-1589.
• Sambrook J, Russell DW (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
• Skurnik D, Ruimy R, Ready D, Ruppe E, Bernède-Bauduin C, Djossou F, Guillemot D, Pier GB, Andremont A (2010) Is exposure to mercury a driving force for the carriage of antibiotic resistance genes?. J Med Microbiol 59:804-807.
• Smit E, Wolters A, van Elsas JD (1998) Self-transmissible mercury resistance plasmids with gene-mobilizing capacity in soil bacterial populations: influence of wheat roots and mercury addition. Appl Environ Microbiol 64:1210-1219.
• Vetriani C, Chew YS, Miller SM, Yagi J, Coombs J, Lutz RA, Barkay T (2005) Mercury adaptation among bacteria from a deep-sea hydrothermal vent. Appl Environ Microbiol 71:220-226.

Publication Dates

• Publication in this collection
  27 Mar 2014
• Date of issue
  Dec 2013

History

• Received
  04 July 2012
• Accepted
  04 Apr 2013