Figures
Abstract
Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6′)-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6′)-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388–16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6′)-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.
Citation: Wen Y, Pu X, Zheng W, Hu G (2016) High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China. PLoS ONE 11(7): e0159418. https://doi.org/10.1371/journal.pone.0159418
Editor: Yi Luo, Nankai University, CHINA
Received: March 3, 2016; Accepted: July 1, 2016; Published: July 18, 2016
Copyright: © 2016 Wen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All nucleotide sequence files are available from the GenBank database (accession numbers:KU644672-KU644712).
Funding: This work was supported by Hangzhou Municipal Bureau of Science and Technology (no. 20140733Q01, http://www.hzst.gov.cn/) and the Health and Family Planning Commission of Zhejiang Province (no. 2015KYA190, http://www.zjwst.gov.cn/). Yanping Wen received the funding mentioned above. Science Foundation of Zhejiang Sci-Tech University (no. 13052168-Y, http://www.zstu.edu.cn), 521 Talent Project of Zhejiang Sci-Tech University (http://www.zstu.edu.cn). Guang Hu received the funding mentioned above. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The dissemination of quinolone-resistant bacterial isolates has become a major problem in infection control worldwide. In Portugal, 23.8% of clinical Escherichia coli isolates were resistant to quinolones [1]. An even higher percentage was reported in the Shanghai area of China, where the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993 [2]. High-level resistance to quinolones is mainly associated with point mutations in the chromosomal genes gyrA and parC, which encode the quinolone targets DNA gyrase and topoisomerase IV, respectively. This chromosome-encoded resistance is transmitted vertically and thus believed to correlate with the phylogenetic lineage of the host. In contrast, plasmid-mediated quinolone resistance (PMQR) confers low-level resistance to quinolones by protection from drug targets and can be transferred horizontally among distantly related lineages [3]. This might favor the emergence of strains with higher resistance to quinolones because of chromosomal mutations. Three major mechanisms are involved in PMQR: (i) limiting quinolone inhibition by Qnr protein protection of drug targets; (ii) modification of the quinolone molecule by the variant aminoglycoside acetyltransferase Aac(6′)-Ib-cr; and (iii) the quinolone-specific efflux pump QepA. Therefore, PMQR might play a role in maintaining resistance levels in bacterial populations in the presence of sub-inhibitory concentrations of antibiotics [4].
Urban rivers provide an ideal setting for the acquisition and spread of PMQR because of continuous pollution by quinolones from anthropogenic sources [5–7], e.g., effluents from municipalities, hospitals, and aquaculture. Effluents from these sources may have a quinolone concentration high enough to exert selective pressure on environmental bacteria [8]. Additionally, effluents from these sources can contain a variety of quinolone-resistant pathogens and mobile genetic elements carrying PMQR genes [6, 9]. Discharge may alter biodiversity in an ecosystem, as well as the characteristic microbiota of water animals. Moreover, since several aquatic bacterial species have intrinsic qnr genes [10, 11], mixing with allochthonous species from different sources is likely to promote genetic exchange, which may exacerbate clinical drug resistance and reduce environmental quality.
There is a growing interest in exploring the emergence of PMQR in bacteria from hospital and aquaculture effluent [6, 9, 12, 13]. However, little information is available on the affected rivers, despite the quality of these rivers having a considerable influence on the health of nearby residents. Recent studies have indicated that wastewater treatment plants had a negligible effect on qnrS removal and resistance elimination [14–17]. The incomplete removal severely affects the receiving river, with qnrS found at higher concentration in downstream waters than in samples collected upstream from a discharge point [15].
The aim of this study was to investigate the prevalence and distribution of PMQR bacteria and their gene determinants in rivers that are likely affected by hospital and aquaculture effluent. The transferability and genetic environments of PMQR genes were also analyzed. Knowledge of the sources and mechanisms of PMQR dissemination in different environments can lead to the development of effective strategies to control antibiotic resistance and assess human health risk.
Materials and Methods
Bacterial Isolates
Since the sampling locations were urban rivers open to the public, and no endangered or protected species were involved, no specific permissions were required for this field study. Water samples were collected from 9 aquatic environments in Hangzhou, China, from November 2014 to October 2015. Of these, 4 were situated near hospitals or medical schools (H1–H4), 4 were near aquaculture (A1–A4), and 1 was water source conversation area and was considered as clean water (C). At each site, representative samples were collected in 500-mL glass bottles in triplicate at 3-month intervals. The particles were filtered through a sterile 0.22-μm membrane and inoculated onto Mueller-Hinton (MH) agar plates supplemented with 5 μg/mL ciprofloxacin. Individual colonies were picked up based on morphology. All experiments in this study were performed in parallel.
Characterization of PMQR Bacteria and Their Gene Determinants
Total DNA was extracted using a simple boiling method. The qnrA, qnrB, and qnrS genes were screened using a multiplex PCR method as described previously [18]. The genes qnrC, qnrD, aac(6′)-Ib-cr, and qepA were also detected [19]. The 16S rRNA, gyrB, and ropD genes were amplified and sequenced for phylogenetic analysis [20].
Transferability of PMQR Genes
Transferability of PMQR genes from the environmental isolates was tested, with E. coli J53 AziR and E. coli DH5α used as recipients for conjugation and transformation experiments [21], respectively. The transconjugants were screened on Luria-Bertani (LB) agar plates supplemented with sodium azide (100 μg/mL) plus ciprofloxacin (0.05 μg/mL). The transformants were selected on LB agar plates with ciprofloxacin (0.05 μg/mL). PCR was performed to identify the PMQR genes acquired by the E. coli transconjugants and transformants, using primers and conditions described elsewhere [18].
Antibiotic Susceptibility Testing
Antibiotic susceptibility to amikacin, ampicillin, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, trimethoprim/sulphamethoxazole, and tetracycline was assayed by the disc diffusion method, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2012). E. coli ATCC 25922 was used as a control strain. The minimal inhibitory concentrations (MICs) of ciprofloxacin were determined using a broth microdilution method in accordance with CLSI guidelines.
Plasmid Analysis
The E. coli transconjugants and transformants were grown overnight at 37°C in LB broth containing ciprofloxacin (0.05 μg/mL). Plasmid DNA was extracted using the Qiagen Plasmid Midi Kit (Qiagen Science Inc., MD, USA). Complete nucleotide sequences were determined using a primer walking strategy. The primers P1-F (5′-AACTCAATACCGTAGCAAT-3′) and P1-R (5′-TTTATGTCACGCCGAACT-3′), which target the qnrS2 gene and read outward, were applied to long-range PCR. Thermal cycling conditions were as follows: 1 min at 94°C; 35 cycles of 15 s at 93°C, 30 s at 55°C, and 7 min at 72°C,followed by a 7 min extension at 72°C. Direct sequencing was carried out using ABI PRISM Big Dye Terminator Cycle Sequencing technology (Applied BioSystems, Foster City, USA). Results were analyzed using the software DNA Sequencing Analysis V5.1 (ABI). A sequence analysis was carried out using BLASTn, BLASTx, DNASTAR Lasergene 8.0, and the open reading frame (ORF) finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The plasmids were named according to their hosts.
Phylogenetic Analysis
Multiple sequence alignments were performed using ClustalW in MEGA 5.10 [22]. Phylogenetic trees were constructed using the neighbor-joining method, and the robustness was evaluated by bootstrap analyses based on 1000 resamplings.
Statistics
Statistical analyses were performed using SPSS 22.0 for Windows (SPSS Inc., Chicago, IL). The prevalence of PMQR genes or antibiotic resistance phenotypes was compared among isolates from different origins or taxonomic groups using the chi-square test at a significance level of 0.05.
Nucleotide Sequence Accession Numbers
Representative 16S rRNA, gyrB, and rpoD gene sequences were deposited in GenBank under accession numbers KU644678 to KU644707. The aac(6′)-Ib, aac(6′)-Ib-cr, and qnr gene sequences were submitted to GenBank with accession numbers KU644708 to KU644712. The complete nucleotide sequences of plasmids were deposited in GenBank under accession numbers KU644672 to KU644677.
Results
Prevalence of PMQR Genes in Aquatic Environments
In total, 188 ciprofloxacin-resistant bacterial isolates were obtained from rivers near hospitals (n = 85), aquaculture (n = 83), or clean water (n = 20). Through PCR-based screening, 56 environmental bacteria (29.8%, 56/188) were found to be PMQR positive (Table 1). qnr genes were detected in 44 isolates (23.4%, 44/188), including qnrB2 in 16, qnrS1 in 3, and qnrS2 in 25 isolates. aac(6′)-Ib-cr genes were detected in 32 isolates (17.0%, 32/188). Other PMQR genes, including qnrA, qnrC, qnrD, and qepA, were not detected.
The prevalence of PMQR in isolates from hospital-associated samples, aquaculture-associated samples, or clean water was 28.3% (24/85), 37.3% (31/83), and 5% (1/20), respectively. The prevalence of qnrS1 and qnrS2 genes was not associated with a specific sampling site (p > 0.05). The aac(6′)-Ib-cr and qnrB2 genes showed a significantly higher prevalence in aquaculture samples than in hospital samples (p < 0.05), but their prevalence was not significantly different between hospital samples and clean water (p > 0.05).
Phylogenetic analyses based on the 16S rRNA, gyrB, and ropD genes showed that PMQR bacteria grouped into two main taxonomic groups, namely Enterobacteriaceae and Aeromonadaceae (Fig 1). The prevalence of some PMQR genes, including qnrB2, qnrS2, and aac(6′)-Ib-cr, was significantly associated with certain taxonomic groups (p < 0.05). Ninety-two percent of qnrS2-carrying strains were identified as belonging to Aeromonadaceae, whereas 100% of qnrB2- and 81.3% of aac(6′)-Ib-cr-carrying strains were identified as Enterobacteriaceae.
Bootstrap values (≥50%) generated from 1000 replicates are indicated at branch points. The sampling sites, PMQR genes, antibiotic resistance, transferability of PMQR genes, and minimal inhibitory concentrations (MICs) of ciprofloxacin are indicated in tables. H1–H4, rivers near hospitals; A1–A4, rivers near aquaculture; C, clean water. AK, amikacin; Amp, ampicillin; C, chloramphenicol; Cf, cephalothin; Cip, ciprofloxacin; E, erythromycin; Na, nalidixic acid; S, streptomycin; Stx, trimethoprim/sulphamethoxazole; Tet, tetracycline.
Antibiotic Susceptibility of Environmental Isolates
All PMQR isolates (n = 56) presented multidrug resistance (MDR), i.e., resistance to antibiotics belonging to three or more classes (Fig 1). The most commonly encountered resistances were to ciprofloxacin (100%, 56/56) and nalidixic acid (100%, 56/56), followed by ampicillin (98.2%, 55/56), cephalothin (94.6%, 53/56), erythromycin (92.9%, 52/56), trimethoprim/sulphamethoxazole (89.3%, 50/56), tetracycline (83.9%, 47/56), chloramphenicol (73.2%, 41/56), streptomycin (73.2%, 41/56), and amikacin (5.4%, 3/56) (Fig 2).
H1–H4, rivers near hospitals; A1–A4, rivers near aquaculture; C, clean water. AK, amikacin; Amp, ampicillin; C, chloramphenicol; Cf, cephalothin; Cip, ciprofloxacin; E, erythromycin; Na, nalidixic acid; S, streptomycin; Stx, trimethoprim/sulphamethoxazole; Tet, tetracycline.
For most antibiotic classes, the prevalence of resistance did not significantly differ (p > 0.05) by sampling site. The exception was the significantly higher (p < 0.05) prevalence of resistance to chloramphenicol, erythromycin, and sulfamethoxazole/trimethoprim in isolates from aquaculture samples. The average MICs of ciprofloxacin against Enterobacteriaceae (82 μg/mL) and Aeromonadaceae (62 μg/mL) were not significantly different (p > 0.05).
Transfer of PMQR Genes
Of the Enterobacteriaceae (n = 29), 4 strains (13.8%) were able to transfer PMQR genes to E. coli DH5α by transformation of [2 aac(6′)-Ib-cr, 1 qnrS1, and 1 qnrS2], but all failed to produce transconjugants. Of the Aeromonadaceae (n = 23), 16 strains (69.6%) were able to transfer PMQR genes to E. coli DH5α by transformation of [5 qnrS2, 9 qnrS2 associated with aac(6’)-Ib, and 2 qnrS2 associated with aac(6′)-Ib-cr]; however, only one transconjugant carrying qnrS2 was obtained (Fig 1).
Overall, the rates of mobile aac(6′)-Ib-cr, qnrB2, qnrS1, and qnrS2 genes were 12.5% (4/32), 0% (0/16), 33.3% (1/3), and 72.0% (18/25), respectively. The presence of PMQR genes increased the MICs of ciprofloxacin two- to eight-fold compared with those in the control E. coli recipients (Fig 1).
Characterization of qnrS2-Harboring Plasmids in Aeromonas spp.
Eleven E. coli transformants derived from clonally unrelated Aeromonas strains were selected for plasmid analysis. These plasmids were small in size (6,388–8,663 bp) and belonged to the IncQ family except for an IncU plasmid, pHP2 (16,197 bp). All plasmids contained qnrS2 inserted at either side of the oriV region (IncQ) or the gene mpR coding for a putative zinc-metalloprotease (IncU) in the form of a mobile insertion cassette (mic). The mic was bracketed by 22-bp imperfect inverted repeats and a 5-bp duplication of the target site (Fig 3), suggesting the acquisition of this structure by transposition. Based on genetic structure, three IncQ plasmid groups were identified.
Open reading frames (ORFs) are shown as arrows indicating the direction of transcription. Inverted repeats (IRL, left inverted repeat; IRR, right inverted repeat) are shown in boxes with black arrows indicating the direction. Their lengths and sequences are shown below the structures. Direct repeats flanking the mobile insertion cassette (mic) are shown above the map. Homologous regions are indicated by gray shading. MITE, miniature inverted transposable element.
The first IncQ plasmid group was isolated from Aeromonas caviae and included two identical plasmids, pHP18 and pAB4, and one variant pHP1. Plasmid pHP18, the sole plasmid found in this study, which was capable of mobilization through conjugation, was 6,388 bp in size with a G+C content of 57%. It contained 6 ORFs: repA and repC genes for plasmid replication, mobA and mobC genes for plasmid mobilization, the qnrS2 gene for quinolone resistance, and a repressor gene (Table 2). The qnrS2 gene was 100% identical with its Aeromonas spp. counterpart. The plasmid backbone showed high similarity with two IncQ-1 plasmids, pCHE-A (66% coverage, 85% identity) and pMS260 (65% coverage, 80% identity). Plasmid pCHE-A originated from Enterobacter cloacae from Canada, and harbored the blaGES-5 gene [11], whereas pMS260 was a streptomycin and sulfonamide resistance-coding plasmid isolated from Actinobacillus pleuropneumoniae in Japan [23]. Despite sharing three common 22-bp iterons with pMS260, the backbone of pHP18 showed characteristics that were distinct from those of other IncQ-family plasmids, including entirely different A+T-rich and G+C-rich regions, and the absence of a highly conserved 15-bp region. A codon bias analysis indicated that the backbone genes of pHP18 had a strong preference for a G or C in the third position (72.8%), while only 39.3% of the codons of qnrS2 had a G or C residue in that position. Compared with pHP18, pHP1 had an additional 22-bp iteron insertion within oriV (Fig 4). Phylogenetic analysis of the replication protein sequences showed that pHP18 and pHP1 grouped with the IncQ-1 family (Fig 5).
Direct repeats are shown by arrows above the sequence. Consensus sequences between plasmids are shown.
Bootstrap values (≥50%) generated from 1000 replicates are indicated at branch points.
The second IncQ plasmid group included three identical plasmids from A. caviae. The representative plasmid pAB5 was 7,212 bp in size, had a G+C content of 60%, and contained 5 ORFs: repA, repC, mobA, mobC, and qnrS2. pAB5 showed a high degree of similarity (99.9%) with the qnrS2-carrying plasmid pAHH04 from A. hydrophila, except for the iteron region [24](Fig 4), and belonged to the IncQ-3 group (Fig 5).
The third IncQ plasmid group was widely distributed among the Aeromonas species, including A. caviae, A. hydrophila, and A. allosaccharophila. This group consisted of three identical plasmids (pHP16, pHP17, and pAB16) and one variant pHP5. Plasmid pHP16 was 8,213 bp in size, had a G+C content of 59%, and belonged to the IncQ-3 group (Fig 5). It contained 6 ORFs: repA, repC, mobA, mobC, qnrS2, and a methyl-accepting chemotaxis protein-encoding gene (orf1). Plasmid pHP16 showed a sequence nearly identical to that of plasmids pHP5, pGNB2, and pBRST7.6 [25, 26], except for a variable region located between qnrS2 and oriV (nt 7367–8013 bp in pHP16) that resulted from different truncation patterns in Tn1721.
The plasmid pHP2, harboring the qnrS2 and aac(6′)-Ib-cr genes in A. caviae, had a typical IncU backbone for plasmid replication and maintenance functions (Fig 3). It was 16,197 bp in size, with an average G+C content of 55%. qnrS2 was inserted into mpR as a mic. aac(6′)-Ib-cr, located on an incomplete class 1 integron, was inserted into the gene nic, encoding VirD2 relaxase. pHP2 was highly related to the PMQR-carrying IncU plasmid pAH6 (98% coverage, 100% identity) and pAC3 (100% coverage, 99% identity) found in Aeromonas spp. from Czech Republic and Korea [27]. The divergence was mainly due to different repetitive sequences surrounding the rep genes. In pHP2, the repetitive sequences located upstream of repA (319 bp) and downstream of repB (739 bp) were organized as (r3r1)3r3 and (r1)8r2, respectively. The corresponding sequences in pAH6 were organized as (r3r1r3)2 and (r1)8r2, whereas in pAC3 the counterparts were organized as (r3r1)3r3 and r1(partial)r1(partial)r1r2. Furthermore, the left inverted repeat of mic in pHP2, as well as a partial truncated region of mpR, was missing in pAH6. This difference may have resulted from plasmid rearrangements subsequent to the acquisition of the qnrS2-carrying mic.
Discussion
The PMQR determinants have been identified in a number of environmental bacteria worldwide. Their prevalence appears to vary considerably depending on the selection criteria of studied strains, ranging from 16.7 to up to 58.0% [9, 28]. In this study, the prevalence of PMQR genes in isolates from rivers was 29.8%, with aac(6′)-Ib-cr and qnrS2 as the predominant genes. This conclusion was supported by previous studies demonstrating the frequent occurrences of these genes in environmental isolates [9, 15, 28]. The qnrS-type genes seem to be the most commonly identified acquired qnr genes in the environment [4]. They have been mainly identified from waterborne species, and in particular Aeromonas spp. [29–31]. aac(6′)-Ib-cr is widespread geographically and stable over time [32]. It has often been more common than qnr alleles. The occurrence of qepA is quite limited among environmental isolates, but in animals might be significant, with a strong association with rmtB genes [33]. Noticeably, some studies identified a high prevalence of other PMQR genes such as qnrD or qnrB in aquatic environments [2, 28]. Factors, such as bacterial species and microbial habitat, may play important roles in the development and spread of antibiotic resistance.
In this study, the prevalence of the genes aac(6′)-Ib-cr and qnrB2 was significantly higher in aquaculture samples than in hospital samples. However, there was no significant difference between hospital samples and clean water. This suggests a site-related dissemination route and a non-clinical origin for the two genes. Rivers near aquaculture may serve as a source of aac(6′)-Ib-cr and qnrB2 genes in the urban water cycle, as indicated by other studies focused on fish farms [34, 35]. A site-related prevalence may be related to a given habitat, where the success of resistance acquisition depends on the fitness of the target bacterium (receivers of horizontal gene transfer) [14]. In agreement with this observation, aac(6′)-Ib-cr and qnrB2 genes were mainly detected in a specific taxonomic group, Enterobacteriaceae. There may be environmental conditions or selective pressures imposed in rivers near aquaculture that contribute to this site-related dissemination pattern.
Unlike aac(6′)-Ib-cr and qnrB2, the prevalence of qnrS2 was not site-related; rather, it was species-related. The fact that qnrS2 was mainly identified from Aeromonas spp., typical inhabitants of aquatic environments, suggested a non-clinical source for this gene in bacteria from urban rivers. This conclusion is supported by previous studies demonstrating the frequent occurrence of qnrS2 in municipal wastewater and rare detection in hospital effluent [9, 14, 16]. The ubiquity and genomic plasticity of Aeromonas spp. may contribute to the homogeneous distribution of qnrS2 in water.
All PMQR bacteria examined showed resistance to more than three classes of antibiotics. In Enterobacteriaceae, multidrug resistance may be related to the high prevalence of aac(6′)-Ib-cr, which confers resistance to kanamycin, tobramycin, netilmicin, amikacin, and ciprofloxacin, and is associated with an MDR phenotype [6, 36]. Moreover, qnr genes have been found to exist in many extended-spectrum beta-lactamase- and AmpC-producing Enterobacteriaceae [37–39], suggesting their co-selection with other resistance elements. In Aeromonadaceae, because of their intrinsic resistance to some beta-lactams and susceptibility to antibiotic resistance acquisition [9], Aeromonas spp. are frequently multidrug resistant [16, 17]. The significantly higher prevalence of resistance to chloramphenicol, erythromycin, and sulfamethoxazole/trimethoprim in isolates from aquaculture samples may be associated with the wide range of antibiotics used in aquaculture [40]. High rates of resistance to the older, heavily used antibiotics (chloramphenicol, sulfonamides, and tetracyclines) have frequently been detected in fish food and fish pathogens such as Aeromonas salmonicida [34, 35, 41].
The qnrS2 genes from Aeromonas spp. were often identified in IncU, IncQ, and ColE plasmids [24, 25, 27, 42]. In this study, IncQ plasmids were highly prevalent (90.9%). The wide distribution of IncQ plasmids carrying PMQR genes can be attributed to several mechanisms, including their ability to readily mobilize and replicate in a broad range of hosts, their low molecular weight that minimizes metabolic load, and their high copy numbers that ensure stability [43]. The ubiquity of Aeromonas species may have facilitated evolution by recombination of IncQ plasmids with a large number of genetic elements or resistance gene insertions.
Among qnrS2-carrying plasmids in Aeromonas spp., pHP18 is the first identified plasmid that can be mobilized by conjugation. However, it did not carry the mating pair formation (Mpf) components essential for efficient transfer by conjugation. A larger conjugative plasmid may be co-resident with pHP18, providing it with the Mpf components. A resistance gene reservoir not only allows resistant genes to stably exist, but also facilitates transfer of these genes to other species in a natural state. Our findings highlighted the important role of Aeromonas species as a PMQR gene reservoir. Plasmid pHP18 shows >80% identity with plasmids from A. pleuropneumoniae and E. cloacae in the backbone region, suggesting a possible origin from bacterial pathogens of humans or animals. The codon bias analysis indicated that qnrS2 was likely inserted after a functional replicon had evolved in IncQ family plasmids.
In addition to the accessory genes, three 22-bp iterons in IncQ plasmids were subject to insertion or mutation, as shown in the pHP18 and pAB5 variants. Iterons were the major incompatibility determinants in IncQ plasmids, so point mutations within the three 20-bp conserved repeats of iterons could result in an inability or significantly reduced ability to exert incompatibility, initiate replication, or transfer genes by conjugation [43]. Meanwhile, the iteron number had a negative effect on plasmid copy number, as well as the antibiotic resistance level, and imposed metabolic burden [44]. Hence, variations in the iteron sequence may reflect the need for balance between high antibiotic resistance and a stable existence [45]. This may explain why pHP18 conferred a higher level of quinolone resistance to E. coli recipients than pHP1 did.
In IncU plasmids, a fragment encompassing repB with r1/r2 repetitive regions was essential for minireplicon functionality [46]. Other repetitive regions seemed to be a hotspot for sequence elimination, as shown in the pHP2 variants. Plasmid pHP2 is one of the smallest IncU plasmids identified so far. Recently, a smaller IncU plasmid pPA-2 (7,995 bp) was identified in Pseudomonas aeruginosa clinical isolates [47]. Both these plasmids have an elimination of conjugative transfer region. Despite loss of conjugative abilities, small IncU plasmids can be efficiently transferred in aquatic environments by transformation, due to the general ability of Aeromonas environmental isolates to acquire free DNA [48].
Overall, this study suggested aquaculture as a possible source of aac(6′)-Ib-cr and qnrB2 in aquatic environments. Enterobacteriaceae, monitored in water quality controls, were important hosts of these two genes. The ubiquitous bacteria, Aeromonas spp., served as vectors for qnrS2 with the help of IncQ-type plasmids. Evidence for water-human transmission of Aeromonas species was available [49, 50], and a qnrS-containing plasmid was identified in an Aeromonas sp. clinical isolate [51]. Given to the patterns of acquired antimicrobial resistance, more attentions should be paid to the antibiotic resistance surveillance of both clinical and environmental Aeromonas isolates.
Author Contributions
Conceived and designed the experiments: YW GH. Performed the experiments: YW XP WZ. Analyzed the data: YW GH XP. Contributed reagents/materials/analysis tools: YW XP WZ GH. Wrote the paper: YW GH XP.
References
- 1. Adriaenssens N, Coenen S, Versporten A, Muller A, Minalu G, Faes C, et al. European Surveillance of Antimicrobial Consumption (ESAC): outpatient quinolone use in Europe (1997–2009). J Antimicrob Chemother. 2011;66 Suppl 6:vi47–56. pmid:22096066
- 2. Zhao JY, Dang H. Coastal seawater bacteria harbor a large reservoir of plasmid-mediated quinolone resistance determinants in Jiaozhou Bay, China. Microb Ecol. 2012;64(1):187–99. pmid:22252223
- 3. Yang H, Duan G, Zhu J, Zhang W, Xi Y, Fan Q. Prevalence and characterisation of plasmid-mediated quinolone resistance and mutations in the gyrase and topoisomerase IV genes among Shigella isolates from Henan, China, between 2001 and 2008. Int J Antimicrob Agents. 2013;42(2):173–7. pmid:23796894
- 4. Poirel L, Cattoir V, Nordmann P. Plasmid-mediated quinolone resistance; interactions between human, animal, and environmental ecologies. Front Microbiol. 2012;3:24. pmid:22347217
- 5. Xia R, Ren Y, Xu H. Identification of plasmid-mediated quinolone resistance qnr genes in multidrug-resistant gram-negative bacteria from hospital wastewaters and receiving waters in the Jinan area, China. Microb Drug Resist. 2013;19(6):446–56. pmid:23844849
- 6. Varela AR, Macedo GN, Nunes OC, Manaia CM. Genetic characterization of fluoroquinolone resistant Escherichia coli from urban streams and municipal and hospital effluents. FEMS Microbiol Ecol. 2015;91(5).
- 7. Alouache S, Estepa V, Messai Y, Ruiz E, Torres C, Bakour R. Characterization of ESBLs and associated quinolone resistance in Escherichia coli and Klebsiella pneumoniae isolates from an urban wastewater treatment plant in Algeria. Microb Drug Resist. 2014;20(1):30–8. pmid:23952363
- 8. Tello A, Austin B, Telfer TC. Selective pressure of antibiotic pollution on bacteria of importance to public health. Environ Health Perspect. 2012;120(8):1100–6. pmid:22571927
- 9. Varela AR, Nunes OC, Manaia CM. Quinolone resistant Aeromonas spp. as carriers and potential tracers of acquired antibiotic resistance in hospital and municipal wastewater. Sci Total Environ. 2016;542(Pt A):665–71. pmid:26546762
- 10. Poirel L, Liard A, Rodriguez-Martinez JM, Nordmann P. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J Antimicrob Chemother. 2005;56(6):1118–21. pmid:16227349
- 11. Poirel L, Rodriguez-Martinez JM, Mammeri H, Liard A, Nordmann P. Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob Agents Chemother. 2005;49(8):3523–5. pmid:16048974
- 12. Chandran SP, Diwan V, Tamhankar AJ, Joseph BV, Rosales-Klintz S, Mundayoor S, et al. Detection of carbapenem resistance genes and cephalosporin, and quinolone resistance genes along with oqxAB gene in Escherichia coli in hospital wastewater: a matter of concern. J Appl Microbiol. 2014;117(4):984–95. pmid:24975198
- 13. Devarajan N, Laffite A, Mulaji CK, Otamonga JP, Mpiana PT, Mubedi JI, et al. Occurrence of antibiotic resistance genes and bacterial markers in a tropical river receiving hospital and urban wastewaters. PLoS One. 2016;11(2):e0149211. pmid:26910062
- 14. Figueira V, Vaz-Moreira I, Silva M, Manaia CM. Diversity and antibiotic resistance of Aeromonas spp. in drinking and waste water treatment plants. Water Res. 2011;45(17):5599–611. pmid:21907383
- 15. Rodriguez-Mozaz S, Chamorro S, Marti E, Huerta B, Gros M, Sanchez-Melsio A, et al. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river. Water Res. 2015;69:234–42. pmid:25482914
- 16. Cisar CR, Henderson SK, Askew ML, Risenhoover HG, McAndrews CR, Kennedy SD, et al. Antibiotic resistance in Aeromonas upstream and downstream of a water resource recovery facility. Water Environ Res. 2014;86(9):835–43. pmid:25327024
- 17. Picao RC, Cardoso JP, Campana EH, Nicoletti AG, Petrolini FV, Assis DM, et al. The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage. Diagn Microbiol Infect Dis. 2013;76(1):80–5. pmid:23478032
- 18. Cattoir V, Poirel L, Rotimi V, Soussy CJ, Nordmann P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother. 2007;60(2):394–7. pmid:17561500
- 19. Vredenburg J, Varela AR, Hasan B, Bertilsson S, Olsen B, Narciso-da-Rocha C, et al. Quinolone-resistant Escherichia coli isolated from birds of prey in Portugal are genetically distinct from those isolated from water environments and gulls in Portugal, Spain and Sweden. Environ Microbiol. 2014;16(4):995–1004. pmid:24034690
- 20. Martinez-Murcia AJ, Monera A, Saavedra MJ, Oncina R, Lopez-Alvarez M, Lara E, et al. Multilocus phylogenetic analysis of the genus Aeromonas. Syst Appl Microbiol. 2011;34(3):189–99. pmid:21353754
- 21. Briales A, Rodriguez-Martinez JM, Velasco C, de Alba PD, Rodriguez-Bano J, Martinez-Martinez L, et al. Prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases in Spain. Int J Antimicrob Agents. 2012;39(5):431–4. pmid:22365240
- 22. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731–9. pmid:21546353
- 23. Ito H, Ishii H, Akiba M. Analysis of the complete nucleotide sequence of an Actinobacillus pleuropneumoniae streptomycin-sulfonamide resistance plasmid, pMS260. Plasmid. 2004;51(1):41–7. pmid:14711528
- 24. Han JE, Kim JH, Choresca CH Jr, Shin SP, Jun JW, Chai JY, et al. A small IncQ-type plasmid carrying the quinolone resistance (qnrS2) gene from Aeromonas hydrophila. Lett Appl Microbiol. 2012;54(4):374–6. pmid:22260532
- 25. Majumdar T, Das B, Bhadra RK, Dam B, Mazumder S. Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish. Plasmid. 2011;66(2):79–84. pmid:21664930
- 26. Bonemann G, Stiens M, Puhler A, Schluter A. Mobilizable IncQ-related plasmid carrying a new quinolone resistance gene, qnrS2, isolated from the bacterial community of a wastewater treatment plant. Antimicrob Agents Chemother. 2006;50(9):3075–80. pmid:16940104
- 27. Dobiasova H, Videnska P, Dolejska M. Complete sequences of IncU plasmids harboring quinolone resistance genes qnrS2 and aac(6')-Ib-cr in Aeromonas spp. from ornamental fish. Antimicrob Agents Chemother. 2015;60(1):653–7. pmid:26525788
- 28. Zhang R, Ichijo T, Huang YL, Cai JC, Zhou HW, Yamaguchi N, et al. High prevalence of qnr and aac(6')-Ib-cr genes in both water-borne environmental bacteria and clinical isolates of Citrobacter freundii in China. Microbes Environ. 2012;27(2):158–63. pmid:22791048
- 29. Chenia HY. Prevalence and characterization of plasmid-mediated quinolone resistance genes in Aeromonas spp. isolated from South African freshwater fish. Int J Food Microbiol. 2016;231:26–32. pmid:27180024
- 30. Cattoir V, Poirel L, Aubert C, Soussy CJ, Nordmann P. Unexpected occurrence of plasmid-mediated quinolone resistance determinants in environmental Aeromonas spp. Emerg Infect Dis. 2008;14(2):231–7. pmid:18258115
- 31. Picao RC, Poirel L, Demarta A, Silva CS, Corvaglia AR, Petrini O, et al. Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake. J Antimicrob Chemother. 2008;62(5):948–50. pmid:18772162
- 32. Jacoby GA, Strahilevitz J, Hooper DC. Plasmid-mediated quinolone resistance. Microbiol Spectr. 2014;2(5).
- 33. Liu BT, Wang XM, Liao XP, Sun J, Zhu HQ, Chen XY, et al. Plasmid-mediated quinolone resistance determinants oqxAB and aac(6')-Ib-cr and extended-spectrum beta-lactamase gene blaCTX-M-24 co-located on the same plasmid in one Escherichia coli strain from China. J Antimicrob Chemother. 2011;66(7):1638–9. pmid:21546384
- 34. Jiang HX, Tang D, Liu YH, Zhang XH, Zeng ZL, Xu L, et al. Prevalence and characteristics of beta-lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China. J Antimicrob Chemother. 2012;67(10):2350–3. pmid:22809702
- 35. Ishida Y, Ahmed AM, Mahfouz NB, Kimura T, El-Khodery SA, Moawad AA, et al. Molecular analysis of antimicrobial resistance in gram-negative bacteria isolated from fish farms in Egypt. J Vet Med Sci. 2010;72(6):727–34. pmid:20145377
- 36. Chen X, Zhang W, Pan W, Yin J, Pan Z, Gao S, et al. Prevalence of qnr, aac(6')-Ib-cr, qepA, and oqxAB in Escherichia coli isolates from humans, animals, and the environment. Antimicrob Agents Chemother. 2012;56(6):3423–7. pmid:22391545
- 37. Galvin S, Boyle F, Hickey P, Vellinga A, Morris D, Cormican M. Enumeration and characterization of antimicrobial-resistant Escherichia coli bacteria in effluent from municipal, hospital, and secondary treatment facility sources. Appl Environ Microbiol. 2010;76(14):4772–9. pmid:20525867
- 38. Fortini D, Fashae K, Garcia-Fernandez A, Villa L, Carattoli A. Plasmid-mediated quinolone resistance and beta-lactamases in Escherichia coli from healthy animals from Nigeria. J Antimicrob Chemother. 2011;66(6):1269–72. pmid:21393162
- 39. Seo MR, Park YS, Pai H. Characteristics of plasmid-mediated quinolone resistance genes in extended-spectrum cephalosporin-resistant isolates of Klebsiella pneumoniae and Escherichia coli in Korea. Chemotherapy. 2010;56(1):46–53. pmid:20203489
- 40. Heuer OE, Kruse H, Grave K, Collignon P, Karunasagar I, Angulo FJ. Human health consequences of use of antimicrobial agents in aquaculture. Clin Infect Dis. 2009;49(8):1248–53. pmid:19772389
- 41. Harnisz M, Korzeniewska E, Golas I. The impact of a freshwater fish farm on the community of tetracycline-resistant bacteria and the structure of tetracycline resistance genes in river water. Chemosphere. 2015;128:134–41. pmid:25698291
- 42. Han JE, Kim JH, Choresca CH Jr, Shin SP, Jun JW, Chai JY, et al. First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene. Lett Appl Microbiol. 2012;55(4):290–4. pmid:22862417
- 43. Loftie-Eaton W, Rawlings DE. Diversity, biology and evolution of IncQ-family plasmids. Plasmid. 2012;67(1):15–34. pmid:22037393
- 44. Loftie-Eaton W, Rawlings DE. Evolutionary competitiveness of two natural variants of the IncQ-like plasmids, pRAS3.1 and pRAS3.2. J Bacteriol. 2010;192(23):6182–90. pmid:20889750
- 45. Meyer R. Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid. 2009;62(2):57–70. pmid:19465049
- 46. Kulinska A, Czeredys M, Hayes F, Jagura-Burdzy G. Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group. Appl Environ Microbiol. 2008;74(13):4119–32. pmid:18502921
- 47. Naas T, Bonnin RA, Cuzon G, Villegas MV, Nordmann P. Complete sequence of two KPC-harbouring plasmids from Pseudomonas aeruginosa. J Antimicrob Chemother. 2013;68(8):1757–62. pmid:23569197
- 48. Huddleston JR, Brokaw JM, Zak JC, Jeter RM. Natural transformation as a mechanism of horizontal gene transfer among environmental Aeromonas species. Syst Appl Microbiol. 2013;36(4):224–34. pmid:23541366
- 49. Khajanchi BK, Fadl AA, Borchardt MA, Berg RL, Horneman AJ, Stemper ME, et al. Distribution of virulence factors and molecular fingerprinting of Aeromonas species isolates from water and clinical samples: suggestive evidence of water-to-human transmission. Appl Environ Microbiol. 2010;76(7):2313–25. pmid:20154106
- 50. Pablos M, Remacha MA, Rodriguez-Calleja JM, Santos JA, Otero A, Garcia-Lopez ML. Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water. Eur J Clin Microbiol Infect Dis. 2010;29(9):1163–72. pmid:20549532
- 51. Sanchez-Cespedes J, Blasco MD, Marti S, Alba V, Alcalde E, Esteve C, et al. Plasmid-mediated QnrS2 determinant from a clinical Aeromonas veronii isolate. Antimicrob Agents Chemother. 2008;52(8):2990–1. pmid:18505860