Antibiotic resistance has become a major public health concern because the organisms that cause infections are becoming less sensitive to antibiotic treatment. Antibiotic resistance traditionally has been studied in bacterial pathogens, limiting efforts to only clinically identified mechanisms . However, antibiotic resistance can also arise in non-pathogenic bacteria as a result of horizontal gene transfer. Moreover, antibiotics are released daily into the natural environment with treated wastewater effluents and through use in animal husbandry. Aquatic bacteria could, therefore, provide a reservoir for antibiotic resistance genes. In this study, we investigated a multidrug-resistant Aeromonas sp. strain P2G1, isolated from the Ter River in Ripoll, Spain.
Strain P2G1 was identified by 16S rRNA gene sequencing (corresponding to positions 27–1492 in the Escherichia coli gene) as Aeromonas sp., with 99.4% sequence similarity to its closest relatives, Aeromonas hydrophila subsp. ranae LMG 19707T and Aeromonas caviae ATCC 15468T. Antibiotic susceptibility tests were determined using the broth microdilution method according to the CLSI guidelines . The strain was resistant to amoxicillin and ciprofloxacin (MICs of >64 μg/mL−1 and 16 μg/mL−1, respectively).
An initial screening, using a multiplex PCR assay for simultaneous detection of qnr genes , showed the presence of a qnrS gene. Subsequently, plasmid DNA extraction was performed using a PureLink Quick plasmid miniprep kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s recommendations. Escherichia coli TOP10 was used as a recipient strain for transformation assays, and transformants were selected on Mueller-Hinton (MH) agar plates containing nalidixic acid (3 μg/mL−1). As expected, no growth was observed on antibiotic control plates when plasmid DNA was not added to the cells. Further analysis identified a single 26.6-kb plasmid (pP2G1) from E. coli transformants, which conferred increased MIC values of several antibiotics (Table 1).
Table 1. Antibiotic susceptibility profiles for Aeromonas sp. strain P2G1 and E. coli TOP10 harbouring natural plasmid pP2G1
| Aeromonas sp. P2G1||>64||<0.06||16||64||32||16||4||16||>128||16||0.5||32||>32|
| E. coli TOP10 + pP2G1||64||<0.06||0.24||4||1||0.72||0.24||0.48||>128||>128||0.25||2||>32|
| E. coli TOP10||4||<0.06||<0.01||1||0.03||<0.01||<0.01||0.01||16||16||0.25||1||<0.1|
The complete nucleotide sequence of pP2G1 was determined using a shotgun sequencing approach (Macrogen, Seoul, Korea). Briefly, randomly sheared plasmid fragments of 3–4 kb were cloned and transformed into E. coli DH10B. Inserts were sequenced by dye terminator chemistry. The sequences were then assembled with phrap (phred/phrap/consed; available from http://www.phrap.org). Nucleotide and amino acid sequence analyses were performed with NCBI (http://www.ncbi.nlm.nih.gov) and European Bioinformatics Institute (http://www.ebi.ac.uk) analysis tools.
The nucleotide sequence of plasmid pP2G1 (EMBL-Bank accession number HE616910) consisted of 26 645 bp in length and belonged to the IncU incompatibility group. Plasmids belonging to this incompatibility group have been isolated from Aeromonas species [4–6], as well as from clinical isolates of E. coli . Annotation of the pP2G1 nucleotide sequence revealed the presence of a qnrS2 gene inserted within the mpR gene encoding a putative zinc-metalloprotease. The nucleotide sequence identified downstream of the qnrS2 gene was identical to that found in qnrS2-positive Aeromonas strains from France and Switzerland [5,6]. However, sequence analysis of the regions flanking the 5′ end of the qnrS2 gene revealed that an IS element, ISKpn9, was present upstream of qnrS2 in strain P2G1 (Fig. 1). This IS element belongs to the ISAs1 family, which has been identified in a Klebsiella pneumoniae clinical strain . Analysis of the ISKpn9 insertion sites revealed a target site duplication (CTATTTTACC) in strain P2G1, in which the qnrS2 gene was plasmid located. This suggests that transposition of ISKpn9 occurs independently of qnrS2 gene acquisition. Moreover, putative promoter sequences were found in the 125-bp sequences that separated the IRL of ISKpn9 from the ATG site of the qnrS2 gene, which suggests that ISKpn9 is not involved in qnrS2 expression.
Figure 1. Genetic map of the multidrug-resistant plasmid pP2G1. Coding regions are shown by arrows indicating the direction of transcription. The two inner circles represent the G+C content plotted against the average G+C content of 50% (black circle) and GC skew information (green and purple circles). The G+C plot was generated by using the CGView tool .
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A class I integron was also identified (Fig. 1), which included four integrated resistance gene cassettes, namely aac(6′)-Ib-cr, blaOXA-1, catB3 and arr-3, encoding an aminoglycoside acetyltransferase, an oxacillinase conferring resistance to β-lactams (penicillins and cephalosporins), an acetyltransferase conferring resistance to chloramphenicol and an ADP-ribosylating transferase conferring resistance to rifampicin, respectively . This class 1 integron showed an identical structure to the In37 integron , and the 3′-conserved segment included the qacEΔ1 and sul1 genes, which provide resistance to quaternary ammonium compounds and sulphonamide, respectively. The In37 integron has been described in an E. coli isolate from China  and in a K. pneumoniae clinical isolate from Argentina . However, the integron identified in plasmid pP2G1 was not associated with an ISCR1 element, as previously reported in those isolates from Argentina and China [9,10]. In addition, it is important to note that aac(6′)-Ib-cr, another plasmid-mediated quinolone determinant, was found in association with the qnrS2 gene on the same plasmid, which may have contributed to reduced ciprofloxacin and norfloxacin susceptibility in both the donor and the transformant strains (Table 1).
Interestingly, a macrolide resistance operon mphA-mrx-mphR was located downstream and in the opposite orientation from an IS element, IS6100. The macrolide-resistance operon encodes a macrolide phosphotransferase (MphA), a protein required for MphA expression (Mrx) and a negative transcriptional regulator (MphR), and confers high-level erythromycin resistance [11,12]. In fact, as a result of the acquisition of the macrolide resistance operon, the susceptibility of E. coli transformants harbouring pP2G1 to erythromycin decreased substantially (Table 1). The macrolide-resistance operon mphA-mrx-mphR and its surrounding regions have been previously found on a K. pneumoniae blaCTX-M-62-encoding plasmid, pJIE137 , and on an E. coli blaCTX-M-15-encoding plasmid, pEK499 .
Antibiotic resistance genes have been detected in several Aeromonas species [5,6,11]. Moreover, those species have been isolated from different sources and geographical areas [15,16]. Thus, our findings contribute to highlighting the role of environmental Aeromonas species as reservoirs of antibiotic resistance genes, which may have important public health implications because of the evolution and emergence of antibiotic resistance genes.