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Characterisation of a class 1 integron associated with the formation of quadruple blaGES-5 cassettes from an IncP-1β group plasmid in Pseudomonas aeruginosa
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Characterisation of a class 1 integron associated with the formation of quadruple blaGES-5 cassettes from an IncP-1β group plasmid in Pseudomonas aeruginosa Teng Xu a,1,2, Jian Wang a,2, Jianchao Ying a, Tingyuan Zhu a, Yabo Liu a, Lei Xu a, Pingping Li a, Peizhen Li a, Jun Ying a, Kewei Li a, Huiguang Yi a, Junwan Lu b, Yunliang Hu a, Tieli Zhou a, Qiyu Bao a,∗ a
Institute of Biomedical Informatics, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Chashan University Town at Ouhai District, Wenzhou 325035, China b School of Medicine, Lishui College, Lishui 323000, China
a r t i c l e
i n f o
Article history: Received 1 November 2017 Accepted 7 July 2018 Available online xxx Keywords: Pseudomonas aeruginosa Carbapenem resistance IncP-1β group plasmid Class 1 integron Gene cassette
a b s t r a c t Integrons are genetic platforms responsible for the dissemination of antimicrobial resistance genes among Gram-negative bacteria, primarily due to their association with transposable elements and conjugative plasmids. In this study, a cassette array containing four identical blaGES-5 genes embedded in a class 1 integron located on an IncP-1β group plasmid from a clinical Pseudomonas aeruginosa strain was identified. Comparative genome analysis and conjugation assay showed that the plasmid pICP-4GES lacked the trbN, trbO and trbP genes but was conjugable. Antimicrobial susceptibility testing revealed that compared with single-copy blaGES-5 complementary strains, both the cloned and chromosome-targeted expression of four copies of blaGES-5 increased the minimum inhibitory concentration (MIC) by one to two dilutions for most of the selected antimicrobials. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that the four consecutive cassettes increased blaGES-5 expression by approximately two-fold compared with the single-copy blaGES-5 strain, suggesting that the level of gene expression was not directly proportional to copy number. In addition, the gene cassette capture assay showed that the global blaGES-5 transfer frequency reached 5.38 × 10–4 . © 2018 Published by Elsevier B.V.
1. Introduction Nosocomial infections caused by carbapenem-resistant Pseudomonas aeruginosa are one of the most common challenges for antimicrobial therapy. Resistance to carbapenems in P. aeruginosa can be attributed to a multitude of intrinsic mechanisms, including various efflux pumps, loss of the porin OprD, and increased AmpC β -lactamase expression [1,2]. In addition to intrinsic mechanisms, acquired mechanisms of resistance, such as metallo-β lactamase and extended-spectrum β -lactamase production, have facilitated the emergence of carbapenem-resistant isolates, which has resulted in limited treatment options during recent decades [3]. Minimum inhibitory concentration (MIC) breakpoints of carbapenems, such as imipenem and meropenem, in P. aeruginosa ∗
Corresponding author. Tel.: +86 577 8668 9779; fax: +86 577 8668 9779. E-mail addresses: [email protected], [email protected] (Q. Bao). 1 Present address: Institute of Translational Medicine, Baotou Central Hospital, Baotou 014040, China. 2 These authors contributed equally to this work.
have been established, and values ≥16 mg/L are defined as clinical resistance [3]. blaGES is a class A carbapenemase-encoding gene, formerly identified as blaIBC-1 [4], and has recently been demonstrated to be widely distributed throughout Europe, South Africa and the Far East in at least five bacterial species, residing as gene cassettes mainly on class 1 integrons [3–6]. In addition, many blaGES cassette-harbouring integrons are located on conjugative plasmids [4–11], laying the molecular basis for rapid spread of a carbapenem-resistant phenotype through horizontal gene transfer. Class 1 integrons are one of the five classes of mobile integrons that are notorious for the dissemination of antimicrobial resistance genes and function as a genetic platform for gene cassette capture and resolution [12–16]. At least 200 different gene cassettes have been identified from class 1 integrons and the mechanisms involved in integrase-mediated gene cassette fixation and excision have been well elucidated [17–23]. Although as many as eight gene cassettes have been found in a single class 1 integron [24], few studies have reported that the same multiple gene cassettes simultaneously appeared in a single integron. In 2016, Chen et al.
https://doi.org/10.1016/j.ijantimicag.2018.07.002 0924-8579/© 2018 Published by Elsevier B.V.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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identified two consecutively identical gene cassettes associated with fosfomycin resistance in a naturally occurring class 1 integron located on a non-conjugative plasmid (pGES-GZ) [25]. Furthermore, different gene cassettes have a distinct transfer frequency, which can be reflected by biased emergence of cassettes in naturally occurring class 1 integrons. In this study, a class 1 integron harbouring four identical blaGES-5 gene cassettes was identified. The resistance activity of this cassette array was characterised both in multi-copy and single-copy states. The gene expression level both in single and quadruple blaGES-5 -harboring strains was also evaluated. Finally, a cassette capture assay was carried out to characterise the transfer efficiency of the blaGES-5 gene cassette. 2. Materials and methods 2.1. Bacterial strains, plasmid elimination and conjugation assay Pseudomonas aeruginosa PA1280 wild-type strain was isolated from a 96-year-old male patient with an upper respiratory tract infection at the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) in 2012. The patient was treated with meropenem prior to isolation of the bacterial strain. The plasmid of P. aeruginosa PA1280 wild-type strain was eliminated (PA1280) using a high-voltage (25 μFD, 200 and 2.5 kV; Bio-Rad, Richmond, CA) electroporation method [26]. The plasmid conjugal transfer experiment was performed with rifampicin-resistant and recA-inactivated Escherichia coli EC600 as the recipient and P. aeruginosa PA1280 as the donor. Overnight cultures (3 mL) from the donor and recipient were mixed and were then harvested and re-suspended in 80 μL of brain–heart infusion (BHI) medium. The mixture was spotted onto a 1 cm2 filter membrane, which was subsequently incubated on a BHI agar plate at 37 °C for 16 h. Bacteria were washed from the membrane, which was further spotted on a Muller–Hinton agar plate containing 10 0 0 μg/mL rifampicin and 100 μg/mL ampicillin to select for pICP-4GESpositive transconjugants. The transconjugants were thus named EC600[pICP-4GES]. 16S rRNA and blaGES-5 gene amplification and sequencing were used to confirm the positive transconjugants. Multilocus sequence typing (MLST) of P. aeruginosa PA1280 wildtype strain was performed using seven housekeeping genes as described previously [27]. 2.2. Plasmid sequencing and analysis The plasmid of PA1280 was extracted using an alkaline lysis method as described elsewhere [28]. A 20-kb library was generated using a SMRTbellTM Template Prep Kit (Pacific Biosciences, Menlo Park, CA) according to the PacBio standard protocol and was sequenced on a PacBio RS II instrument (Pacific Biosciences). In addition, an Illumina library with 300-bp insert sizes was also constructed and was sequenced from both ends using a HiSeq 2500 platform (Illumina Inc., San Diego, CA). The PacBio long reads were initially assembled using Canu software [29]. The Illumina reads were subjected to adaptor trimming and quality filtering and were further mapped onto the primary assembly to identify the lowquality bases and false ‘insertions and deletions’ generated by the primary assembly using Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit [30,31]. A custom-derived script was used to extract the consensus sequence. Potential open reading frames (ORFs) were predicted using Glimmer software [32] and were annotated against the non-redundant (Nr) protein database using the BLASTX program. Comparative genome analysis of pICP-4GES with other IncP-1β group plasmids was performed by BLASTN program and custom-derived Biopython scripts. The complete nucleotide sequence of pICP-4GES has been submitted to NCBI under accession no. MH053445.
2.3. Molecular cloning and gene knock-in assay Single and four blaGES-5 complete ORFs alone with the integron Pc promoter were amplified using P. aeruginosa PA1280 DNA as a template. The PCR primer pairs used are listed in Supplementary Text S1. PCR products containing one and four blaGES-5 genes were digested with EcoRI–BamHI and EcoRI–XbaI, respectively, and were ligated into the same sites on a pUCP24 vector (gentamicin-resistant), resulting in pUCP24::blaGES-5 and pUCP24::4blaGES-5 . Both constructed vectors were transformed into the plasmid-eliminated P. aeruginosa PA1280 (abbreviated as PA1280) and ampG-deleted P. aeruginosa PAO1 (abbreviated as PAO1) strains [33], respectively, forming the transformants named PA1280[pUCP24::blaGES-5 ], PA1280[pUCP24::4blaGES-5 ], PAO1[pUCP24::blaGES-5 ] and PAO1[pUCP24::4blaGES-5 ]. Single or quadruple blaGES-5 was cloned into mini-Tn7 using a one-step cloning method. The recombinant mini-Tn7 vector and the helper plasmid pTNS were co-transformed into PAO1 according to the protocol reported by Choi and Schweizer [34]. Bacterial strains with chromosome-targeted blaGES-5 and 4blaGES-5 were validated by PCR and Sanger sequencing.
2.4. Antimicrobial susceptibility testing In total, 12 bacterial strains were selected for susceptibility testing against 15 antimicrobial agents, including carbapenems, using a standard agar dilution method as follows: ampicillin; piperacillin; piperacillin/tazobactam; cefoxitin; ceftriaxone; cefotaxime; ceftazidime; cefepime; cefoperazone; cefoperazone/sulbactam; aztreonam; imipenem; meropenem; kanamycin; and gentamicin. The results were interpreted according to Clinical and Laboratory Standard Institute (CLSI) 2015 recommendations. Pseudomonas aeruginosa ATCC 27853 was used as the reference strain for quality control.
2.5. Cassette stability test A purified clone of transconjugant EC600[pICP-4GES] was inoculated in 5 mL of lysogeny broth (LB) medium without any antibiotics at 37 °C and shaking at 240 rpm overnight. Then, 10-μL cultures were transferred to 5 mL of fresh LB medium for a second round of cultivation. After 20 similar rounds of culture, the bacterial cultures were diluted and were spread on LB agar. A total of 100 clones were randomly picked and were used as templates for PCR amplification to test the stability of the cassettes.
2.6. Gene expression analysis Overnight bacterial cultures were subcultured in LB medium and were further grown to an optical density at 600 nm (OD600 ) of 1.0. Total RNA from each strain was extracted using an RNAprep Pure Cell/Bacteria Kit with on-column DNase I digestion (TIANGEN Biotech, Beijing, China). RNA was further treated by DNase I and was purified using an RNAclean Kit (TIANGEN Biotech). RNA concentration and purity were evaluated with a NanoDropTM 20 0 0 spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was verified by denaturing agarose gel electrophoresis. cDNA was synthesised using a Super RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time reverse transcription PCR (RT-qPCR) was carried out using AceQ® qPCR SYBR® Green Master Mix (Vazyme Biotech, Nanjing, China). The 30S rRNA rpsL gene was used as an internal control to normalise gene expression quantities between samples. Primer sequences are listed in Supplementary Text S1.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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>1024 512 256 >128 >128 64 128 32 >128 >128 4 64 >256 16 256 >1024 128 64 >128 >128 32 32 16 >128 128 2 32 256 16 256 2 2 2 1 <0.125 <0.125 <0.25 <0.125 <0.125 <0.125 <0.125 <0.125 <0.125 <8 <8
[pUCP24::4blaGES-5 ] [pUCP24::blaGES-5 ]
1024 2 2 >128 8 4 1 1 2 2 1 4 1 32 <8
PA1280
>1024 1024 256 >128 >128 >128 32 64 >128 >128 16 32 256 64 <8
agent
AMP PIP TZP FOX CRO CTX CAZ FEP CFP CSL ATM IPM MEM KAN GEN
EC600 [pICP-4GES]
PA1280 MIC (μg/mL)
SMRT sequencing technology was applied to determine the complete nucleotide sequence of the conjugative plasmid. In addition, an Illumina genomic library was constructed and sequenced and was subsequently used to correct the preliminary assembly generated by the SMRT long reads. The results showed that P. aeruginosa PA1280 harboured a 50 914-bp circular plasmid containing a set of genes responsible for plasmid replication, partition, stable inheritance and conjugal transfer (Fig. 1A). The replication region (ssb and trfA) of the plasmid shared >99% identity with
Antimicrobial
3.2. Plasmid sequencing and comparative genome analysis
Table 1 Antimicrobial susceptibility testing of 12 strains.
Recently, we isolated a P. aeruginosa clinical strain, designated as PA1280, that was resistant to multiple β -lactam antibiotics, including carbapenems, and possessed particularly strong resistance to meropenem compared with commonly isolated carbapenemresistant strains. MLST showed that PA1280 belonged to ST1119. Plasmid extraction and agarose gel electrophoresis exhibited positive results. To address the concerns of acquired carbapenem resistance, the plasmid was eliminated through high-voltage electroporation, which significantly reduced the resistance activity against the selected β -lactams except for cefoxitin (Table 1). Conjugal transfer of the plasmid to E. coli EC600 enhanced the resistance of the transconjugants to a series of β -lactams, including piperacillin, cefoxitin and ampicillin, but not carbapenems, which is consistent with the observations from the resistance profiles of IBC-2 in E. coli [35], and the transconjugants’ susceptibility to carbapenem can be ascribed to the distinct mechanisms of antimicrobial resistance between P. aeruginosa and E.coli. These results indicated that the plasmid from P. aeruginosa PA1280 was conjugative and carried strong resistance determinants against carbapenems, which prompted us to determine its complete sequence.
EC600
3.1. Identification of a conjugative plasmid
512 16 8 32 0.5 <0.125 2 <0.125 <0.125 <0.125 <0.125 0.25 <0.125 <8 <8
PA1280 PA1280
3. Results and discussion
3 MIC, minimum inhibitory concentration; AMP, ampicillin; PIP, piperacillin; TZP, piperacillin/tazobactam; FOX, cefoxitin; CRO, ceftriaxone; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CFP, cefoperazone; CSL, cefoperazone/sulbactam; ATM, aztreonam; IPM, imipenem; MEM, meropenem; KAN, kanamycin; GEN, gentamicin.
512 32 8 64 16 16 4 4 32 16 4 1 16 64 64 128 8 4 64 8 8 2 2 16 4 2 0.5 4 64 64 >1024 512 256 >128 >128 64 32 32 >128 >128 8 4 >256 64 256 32 4 4 128 4 8 1 2 4 2 4 0.25 1 32 <8
>1024 256 64 >128 128 32 32 16 >128 128 4 2 128 64 256
PAO1PAO1-
blaGES-5 [pUCP24::4blaGES-5 ]
PAO1 PAO1 PAO1
[pUCP24::blaGES-5 ]
A class 1 integron with the gene arrangement intI1–blaCARB-2 – aadA2–cmlA1–aadA1 was stored in our laboratory and was used as template for PCR amplification. The fragment intI1–blaCARB-2 , which contains the intact intI1 and truncated blaCARB-2 (61bp coding sequence), was amplified and was cloned into the pUCP24 vector by EcoRI–BamHI digestion. The recombinant vector (pUCP24::intI1–blaCARB-2 , also named as Construct I) was transformed into EC600[pICP-4GES] by electroporation (25 μFD, 200 and 2.5 kV) and was selected on a compound LB agar plate containing 20 μg/mL gentamicin. Transformants were subjected to overnight culture and total plasmid extraction and were further chemically transformed into the E. coli DH5α recipient. Transformants that only harboured the blaGES-5 cassette-captured recombinant vector were screened using 100 μg/mL ampicillin, whereas transformants only harbouring Construct I without a cassette insertion were filtered. The original pICP-4GES could not be efficiently transformed into the E. coli DH5α recipient owing to its larger size. The same experimental procedure was also performed using the original pUCP24 for cassette capture. PCR and Sanger sequencing were used to validate the recombinant vector with the cassette insertion. The recombination frequency from Construct I to Recombinants I (blaGES-5 cassette integrated into attI1-I) and II (blaGES-5 cassette integrated into attI1-II) was calculated as the CFU/mL of the transformants selected by 100 μg/mL ampicillin divided by the CFU/mL of the transformants selected by 20 μg/mL gentamicin. Statistical analysis of the recombination frequency of two attI1 sites was performed by using two independent samples t-test.
4blaGES-5
P. aeruginosa ATCC 27853
2.7. Cassette capture assay
1024 2 2 >128 8 4 1 2 4 2 4 16 <0.125 512 16
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Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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Fig. 1. Genomic structure and comparative analysis of plasmid pICP-4GES from Pseudomonas aeruginosa PA1280. (A) Genomic structure of the conjugative plasmid pICP-4GES. Genes are denoted by arrows and are coloured based on the gene function classification. IRL and IRR represent left and right inverted repeats, respectively. (B) Comparative genome analysis of pICP-4GES with three other IncP-1β group plasmids. Orthologous genes are connected by lines and colour coded, except genes marked with grey colour. Genes labelled with hp represented hypothetical genes.
those well-documented plasmids belonging to the IncP-1β incompatibility group with broad host range, including R751, pB8 and pA22732-IMP [36–38]. Comparative genome analysis showed that the plasmid isolated from P. aeruginosa PA1280 exhibited highly global genomic synteny and possessed a backbone similar to the aforementioned IncP-1β plasmids, whilst the gene content and organisation in the two accessory regions were variable (Fig. 1B). One accessory region of the plasmid was horizontally transferred by a typical Tn3 family transposon identified by a pair of imperfect 35-bp inverted repeated (IRs) immediately flanking the region encoding from tnpA to lysR (Fig. 1A). Unexpectedly, the other accessory region included a class 1 integron harbouring four consecutive blaGES-5 gene cassettes. This class 1 integron contained the complete 5 -conserved segment (CS) but lacked the entire 3 -CS; the truncated trbN gene only encoded the first 30 amino acids of TrbN. Consideration of the above properties, this plasmid was named pICP-4GES. In addition, the coding orientation of the class 1 integron from pICP-4GES was different from pB8 and R751 but was the same as that of pA22732-IMP. Interestingly, compared with R751, pICP-4GES lacked the entire tni region at one end of the class 1 integron, and the trbO and trbP genes were also absent in contrast to the three other plasmids, suggesting that these three genes are not required for conjugation. Multiple identical gene cassettes captured by IntI1 appear to emerge rarely, although as many as eight gene cassettes have been found in a single class 1 integron [24]. To validate this genomic architecture, we first used a pair of blaGES-5 outward primers against P. aeruginosa PA1280 for PCR amplification. In addition to a bright band, there were obscure bands corresponding to the estimated sizes of amplicons spanning two (cassette 1–3 or 2–4) and three (cassette 1–4) cassettes in P. aeruginosa PA1280 (Supplementary Fig. S1). Similar results were observed using a pair of primers for amplification of the blaGES-5 complete ORF (Supplementary Fig. S1). Second, we realigned the Illumina reads to pICP-4GES. The results showed that the average sequencing depth at a one base resolution of the tandem blaGES-5 cassette region was 2684.14, which was ca. 4.00-fold greater than that of the remaining region (670.65) of the plasmid. Third, PCR amplification from the intI1 internal region to the region immediately downstream of the last cassette showed the expected product size, and Sanger sequencing from both ends extended into the blaGES-5 cassette, which supported the SMRT reads assembly. Thus, pICP-4GES harboured a class 1 integron with four copies of the blaGES-5 gene cassette.
3.3. Cassette stability Compared with other IncP-1β group plasmids, the terminal region of this class 1 integron appeared atypical. To evaluate the stability of four identical cassettes after the plasmid was transferred to the recipient, 20 rounds of culture of the transconjugant were performed in the absence of any antibiotics. A total of 100 randomly selected clones were used as template for PCR amplification of the four complete cassettes. The product sizes generated from all the clones were completely equal to that of the wild-type strain, indicating that the cassettes in the recipient strain were stable without undergoing major cassette copy changes. 3.4. Resistance activity differences between quadruple and single blaGES-5 A promoter, termed Pc, is located within the intI1 of class 1 integrons and initiates gene cassette expression [39]. A series of Pc variants have been identified and their activities associated with gene cassette transcription and recombination have been characterised [40,41]. The Pc sequence of the class 1 integron from pICP-4GES was analysed and the results showed that the promoter of the cassette belonged to PcW (weak Pc) (Table 2). To characterise the activity of four consecutive blaGES-5 gene cassettes associated with resistance to antimicrobials, both single and four blaGES-5 cassettes, including PcW, were cloned in the multiple copy vector pUCP24 and were further transformed into two recipients, PA1280 and PAO1, the latter being a standard P. aeruginosa strain whose ampG has been knockedout [33]. Compared with wild-type P. aeruginosa PA1280, both pUCP24::blaGES-5 and pUCP24::4blaGES-5 could fully complement or even enhance the resistance activity of PA1280 to several β -lactams, including meropenem, imipenem and ceftazidime, but not to piperacillin, cefotaxime, cefepime or aztreonam. The incomplete complementation of the resistance to these antimicrobials may be ascribed to another β -lactamase gene, blaP, located on the accessory region of pICP-4GES in the wild-type strain (Fig. 1A). Furthermore, pUCP24::blaGES-5 and pUCP24::4blaGES-5 also significantly enhanced the MICs of all selected β -lactams compared with PAO1, except for aztreonam. The contributions to β -lactam resistance by pUCP24::4blaGES-5 were generally higher than those of pUCP24::blaGES-5 for at least one MIC dilution both in PA1280 and PAO1 strains.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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Table 2 Variants of gene cassette promoters involved in three class 1 integrons. Integron
Promoter Pc
intI1–4blaGES-5 intI1–blaPSE-1 –intI1–aacC1
Pc variant
–35 region
–10 region
TGGACA TGGACA TTGACA
TAAGCT TAAGCT TAAACT
PcW PcW PcS
P2
P2 variant
–35 region
–10 region
TTGTTA TTGTTA TTGTTA
TACAGT TACAGT TACAGT
P2 P2 P2
PcW, weak Pc; PcS, strong Pc.
Fig. 2. Schematic representation of the blaGES-5 gene cassette capture assay. (A) Genes are denoted by coloured arrows. Red circle represents the circular cassette excised from the donor plasmid and subsequently fixed into the corresponding attI1 site. Small paired red arrows indicate the relative positions of the PCR primers used for the cassette capture validation, whereas red–white chimeric arrows represent the relative position of the sequencing primers used for validation. (B) The nucleotide sequences of each original and new generated attI1 and attC sites. Red dashed boxes indicate the attI1 sites that were integrated with the gene cassettes. (C) Recombination frequency between blaGES-5 gene cassette and two different attI1 sites.
Because of the multi-copy status of pUCP24 in the host cell, to monitor and compare the resistance activity of either four or one blaGES-5 in a single copy state, the expression of both 4blaGES-5 and blaGES-5 downstream of the glmS gene in the PAO1 chromosome was targeted using a mini-Tn7 system [34]. Compared with PAO1, chromosome-targeted expression both of 4blaGES-5 and blaGES-5 significantly enhanced the resistance against selected β -lactams. In addition, compared with PAO1-blaGES-5 , PAO14blaGES-5 possessed a one to two MIC dilution higher resistance capacity against all the selected β -lactams except for cefoxitin.
3.5. Gene expression analysis of quadruple and single blaGES-5 cassettes Since the resistance activity of four copies of blaGES-5 was one to two dilution MICs higher than the single blaGES-5 -harboring strain, we wanted to identify the gene expression differences between them. Gene expression analysis of four bacterial strains, including wild-type PA1280, PA1280, PA1280[pUCP24::blaGES-5 ] and PA1280[pUCP24::4blaGES-5 ] was carried out by RT-qPCR during bacterial exponential-growth phase. Compared with
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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PA1280[pUCP24::blaGES-5 ],
PA1280[pUCP24::4blaGES-5 ] increased blaGES-5 expression by 2.07-fold, indicating that the gene expression difference of blaGES-5 between two complementary strains was not directly correlated with the variation in gene copy number. This may have been because the strength of gene expression declines as cassettes become more distant from the promoter [39] and because of the non-additive nature of the contribution to MIC of antimicrobials by multiple identical determinants. In addition, gene expression of blaGES-5 in PA1280[pUCP24::blaGES-5 ] was 2.13-fold higher than P. aeruginosa PA1280 wild-type strain, which could be primarily attributed to the higher copy number of pUCP24 in the bacterial cell. However, blaGES-5 expression in PA1280 was at an undetectable level.
that four copies of blaGES-5 enhanced expression by only approximately two-fold compared with the single-copy strain that was driven by a weak cassette promoter. Funding This work was financially supported by the National Natural Science Foundation of China [grant nos. 81501780, 31500109, 81401702 and 81501808]; the Natural Science Foundation of Zhejiang Province, China [grant no. LQ17H190 0 01]; the Nucleic Acids Based Molecular Diagnostics Innovative Discipline, Health and Family Planning Commission of Zhejiang Province; and start-up funds from the Wenzhou Medical University to TX [grant no. QTJ14002].
3.6. Cassette transfer frequency The blaGES-5 gene cassette is widely distributed in naturally occurring class 1 integrons. In order to characterise the potential transfer efficiency of this cassette, a multi-copy vector pUCP24 was used for potential cassette capture assay, which could provide more opportunities for cassette attachment. In pICP-4GES, only the same cassette emerged, which provided a naturally simple cassette donor for quantification of the blaGES-5 recombination frequency and was thus used as cassette donor plasmid. In addition, a DNA fragment containing intact intI1 together with a truncated blaCARB-2 gene (intI1–blaCARB-2 ) was cloned into the pUCP24 vector (Fig. 2). The modified pUCP24 therefore contains two attI1 sites for potential cassette capture (Fig. 2, Construct I). One is an inherent attI1 site (attI1-II) and the other (attI1-I) is derived from the exogenous fragment. If the vectors capture a cassette by either attI1 site after their transformation into the donor plasmidcontaining strain, the two novel junctions formed by the cassette integration can be amplified by specific PCR primer pairs, which could result in products with an enlarged size in response to the non-cassette-capture vector and would not produce an amplicon with the cassette-donor plasmid (pICP-4GES) alone. Sequence analysis indicated that the Pc variant in intI1–blaCARB-2 was a weak promoter that is identical to that in pICP-4GES, whereas Pc identified from intI1–aacC1 was a strong one (Table 2). The original pUCP24 was transformed into EC600[pICP-4GES] for potential cassette capture. There was no cassette inserted into the inherent attI1-II site of original pUCP24, which was reflected by the phenotypic failure to be selected by the corresponding antimicrobial. However, the pUCP24::intI1–blaCARB-2 (Construct I) was transformed into transconjugants and the re-transformants exhibited the ampicillin-resistant phenotype, indicating that cassettes were fixed into the attachment sites. PCR and sequencing of the selected clones showed that both of the attachment sites were integrated by the blaGES-5 gene cassette. In addition, no cassette-donor plasmid could be detected in any re-transformants. We also did not detect a multiple tandem cassette inserted in any of the two attI1 sites. The relatively global recombination frequency of the cassette capture is ca. 5.38 × 10–4 . The attI1-II × attC recombination (Recombinant II) frequency was 5.04 × 10–4 , which was ca. 15-fold higher than that of the attI1-I × attC recombination (3.45 × 10–5 , Recombinant I; P < 0.001) (Fig. 2). This recombination frequency was ca. 3-fold lower than that of aadA2 cassette (1.87 × 10–4 ) that has been demonstrated to be extensively distributed in naturally occurring class 1 integrons [42]. In conclusion, this study characterised an unusual class 1 integron harbouring four copies of blaGES-5 . The atypical termination at the 3 -CS of the integron did not appear to affect the stability of the copy number of cassettes. The resistance activity of the four blaGES-5 genes was generally stronger than a single determinant with an approximately one to two dilution MIC of the selected β -lactam antibiotics. Gene expression analysis also showed
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Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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International Journal of Antimicrobial Agents 0 0 0 (2018) 1–7
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International Journal of Antimicrobial Agents journal homepage: www.elsevier.com/locate/ijantimicag
Characterisation of a class 1 integron associated with the formation of quadruple blaGES-5 cassettes from an IncP-1β group plasmid in Pseudomonas aeruginosa Teng Xu a,1,2, Jian Wang a,2, Jianchao Ying a, Tingyuan Zhu a, Yabo Liu a, Lei Xu a, Pingping Li a, Peizhen Li a, Jun Ying a, Kewei Li a, Huiguang Yi a, Junwan Lu b, Yunliang Hu a, Tieli Zhou a, Qiyu Bao a,∗ a
Institute of Biomedical Informatics, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Chashan University Town at Ouhai District, Wenzhou 325035, China b School of Medicine, Lishui College, Lishui 323000, China
a r t i c l e
i n f o
Article history: Received 1 November 2017 Accepted 7 July 2018 Available online xxx Keywords: Pseudomonas aeruginosa Carbapenem resistance IncP-1β group plasmid Class 1 integron Gene cassette
a b s t r a c t Integrons are genetic platforms responsible for the dissemination of antimicrobial resistance genes among Gram-negative bacteria, primarily due to their association with transposable elements and conjugative plasmids. In this study, a cassette array containing four identical blaGES-5 genes embedded in a class 1 integron located on an IncP-1β group plasmid from a clinical Pseudomonas aeruginosa strain was identified. Comparative genome analysis and conjugation assay showed that the plasmid pICP-4GES lacked the trbN, trbO and trbP genes but was conjugable. Antimicrobial susceptibility testing revealed that compared with single-copy blaGES-5 complementary strains, both the cloned and chromosome-targeted expression of four copies of blaGES-5 increased the minimum inhibitory concentration (MIC) by one to two dilutions for most of the selected antimicrobials. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that the four consecutive cassettes increased blaGES-5 expression by approximately two-fold compared with the single-copy blaGES-5 strain, suggesting that the level of gene expression was not directly proportional to copy number. In addition, the gene cassette capture assay showed that the global blaGES-5 transfer frequency reached 5.38 × 10–4 . © 2018 Published by Elsevier B.V.
1. Introduction Nosocomial infections caused by carbapenem-resistant Pseudomonas aeruginosa are one of the most common challenges for antimicrobial therapy. Resistance to carbapenems in P. aeruginosa can be attributed to a multitude of intrinsic mechanisms, including various efflux pumps, loss of the porin OprD, and increased AmpC β -lactamase expression [1,2]. In addition to intrinsic mechanisms, acquired mechanisms of resistance, such as metallo-β lactamase and extended-spectrum β -lactamase production, have facilitated the emergence of carbapenem-resistant isolates, which has resulted in limited treatment options during recent decades [3]. Minimum inhibitory concentration (MIC) breakpoints of carbapenems, such as imipenem and meropenem, in P. aeruginosa ∗
Corresponding author. Tel.: +86 577 8668 9779; fax: +86 577 8668 9779. E-mail addresses: [email protected], [email protected] (Q. Bao). 1 Present address: Institute of Translational Medicine, Baotou Central Hospital, Baotou 014040, China. 2 These authors contributed equally to this work.
have been established, and values ≥16 mg/L are defined as clinical resistance [3]. blaGES is a class A carbapenemase-encoding gene, formerly identified as blaIBC-1 [4], and has recently been demonstrated to be widely distributed throughout Europe, South Africa and the Far East in at least five bacterial species, residing as gene cassettes mainly on class 1 integrons [3–6]. In addition, many blaGES cassette-harbouring integrons are located on conjugative plasmids [4–11], laying the molecular basis for rapid spread of a carbapenem-resistant phenotype through horizontal gene transfer. Class 1 integrons are one of the five classes of mobile integrons that are notorious for the dissemination of antimicrobial resistance genes and function as a genetic platform for gene cassette capture and resolution [12–16]. At least 200 different gene cassettes have been identified from class 1 integrons and the mechanisms involved in integrase-mediated gene cassette fixation and excision have been well elucidated [17–23]. Although as many as eight gene cassettes have been found in a single class 1 integron [24], few studies have reported that the same multiple gene cassettes simultaneously appeared in a single integron. In 2016, Chen et al.
https://doi.org/10.1016/j.ijantimicag.2018.07.002 0924-8579/© 2018 Published by Elsevier B.V.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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identified two consecutively identical gene cassettes associated with fosfomycin resistance in a naturally occurring class 1 integron located on a non-conjugative plasmid (pGES-GZ) [25]. Furthermore, different gene cassettes have a distinct transfer frequency, which can be reflected by biased emergence of cassettes in naturally occurring class 1 integrons. In this study, a class 1 integron harbouring four identical blaGES-5 gene cassettes was identified. The resistance activity of this cassette array was characterised both in multi-copy and single-copy states. The gene expression level both in single and quadruple blaGES-5 -harboring strains was also evaluated. Finally, a cassette capture assay was carried out to characterise the transfer efficiency of the blaGES-5 gene cassette. 2. Materials and methods 2.1. Bacterial strains, plasmid elimination and conjugation assay Pseudomonas aeruginosa PA1280 wild-type strain was isolated from a 96-year-old male patient with an upper respiratory tract infection at the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) in 2012. The patient was treated with meropenem prior to isolation of the bacterial strain. The plasmid of P. aeruginosa PA1280 wild-type strain was eliminated (PA1280) using a high-voltage (25 μFD, 200 and 2.5 kV; Bio-Rad, Richmond, CA) electroporation method [26]. The plasmid conjugal transfer experiment was performed with rifampicin-resistant and recA-inactivated Escherichia coli EC600 as the recipient and P. aeruginosa PA1280 as the donor. Overnight cultures (3 mL) from the donor and recipient were mixed and were then harvested and re-suspended in 80 μL of brain–heart infusion (BHI) medium. The mixture was spotted onto a 1 cm2 filter membrane, which was subsequently incubated on a BHI agar plate at 37 °C for 16 h. Bacteria were washed from the membrane, which was further spotted on a Muller–Hinton agar plate containing 10 0 0 μg/mL rifampicin and 100 μg/mL ampicillin to select for pICP-4GESpositive transconjugants. The transconjugants were thus named EC600[pICP-4GES]. 16S rRNA and blaGES-5 gene amplification and sequencing were used to confirm the positive transconjugants. Multilocus sequence typing (MLST) of P. aeruginosa PA1280 wildtype strain was performed using seven housekeeping genes as described previously [27]. 2.2. Plasmid sequencing and analysis The plasmid of PA1280 was extracted using an alkaline lysis method as described elsewhere [28]. A 20-kb library was generated using a SMRTbellTM Template Prep Kit (Pacific Biosciences, Menlo Park, CA) according to the PacBio standard protocol and was sequenced on a PacBio RS II instrument (Pacific Biosciences). In addition, an Illumina library with 300-bp insert sizes was also constructed and was sequenced from both ends using a HiSeq 2500 platform (Illumina Inc., San Diego, CA). The PacBio long reads were initially assembled using Canu software [29]. The Illumina reads were subjected to adaptor trimming and quality filtering and were further mapped onto the primary assembly to identify the lowquality bases and false ‘insertions and deletions’ generated by the primary assembly using Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit [30,31]. A custom-derived script was used to extract the consensus sequence. Potential open reading frames (ORFs) were predicted using Glimmer software [32] and were annotated against the non-redundant (Nr) protein database using the BLASTX program. Comparative genome analysis of pICP-4GES with other IncP-1β group plasmids was performed by BLASTN program and custom-derived Biopython scripts. The complete nucleotide sequence of pICP-4GES has been submitted to NCBI under accession no. MH053445.
2.3. Molecular cloning and gene knock-in assay Single and four blaGES-5 complete ORFs alone with the integron Pc promoter were amplified using P. aeruginosa PA1280 DNA as a template. The PCR primer pairs used are listed in Supplementary Text S1. PCR products containing one and four blaGES-5 genes were digested with EcoRI–BamHI and EcoRI–XbaI, respectively, and were ligated into the same sites on a pUCP24 vector (gentamicin-resistant), resulting in pUCP24::blaGES-5 and pUCP24::4blaGES-5 . Both constructed vectors were transformed into the plasmid-eliminated P. aeruginosa PA1280 (abbreviated as PA1280) and ampG-deleted P. aeruginosa PAO1 (abbreviated as PAO1) strains [33], respectively, forming the transformants named PA1280[pUCP24::blaGES-5 ], PA1280[pUCP24::4blaGES-5 ], PAO1[pUCP24::blaGES-5 ] and PAO1[pUCP24::4blaGES-5 ]. Single or quadruple blaGES-5 was cloned into mini-Tn7 using a one-step cloning method. The recombinant mini-Tn7 vector and the helper plasmid pTNS were co-transformed into PAO1 according to the protocol reported by Choi and Schweizer [34]. Bacterial strains with chromosome-targeted blaGES-5 and 4blaGES-5 were validated by PCR and Sanger sequencing.
2.4. Antimicrobial susceptibility testing In total, 12 bacterial strains were selected for susceptibility testing against 15 antimicrobial agents, including carbapenems, using a standard agar dilution method as follows: ampicillin; piperacillin; piperacillin/tazobactam; cefoxitin; ceftriaxone; cefotaxime; ceftazidime; cefepime; cefoperazone; cefoperazone/sulbactam; aztreonam; imipenem; meropenem; kanamycin; and gentamicin. The results were interpreted according to Clinical and Laboratory Standard Institute (CLSI) 2015 recommendations. Pseudomonas aeruginosa ATCC 27853 was used as the reference strain for quality control.
2.5. Cassette stability test A purified clone of transconjugant EC600[pICP-4GES] was inoculated in 5 mL of lysogeny broth (LB) medium without any antibiotics at 37 °C and shaking at 240 rpm overnight. Then, 10-μL cultures were transferred to 5 mL of fresh LB medium for a second round of cultivation. After 20 similar rounds of culture, the bacterial cultures were diluted and were spread on LB agar. A total of 100 clones were randomly picked and were used as templates for PCR amplification to test the stability of the cassettes.
2.6. Gene expression analysis Overnight bacterial cultures were subcultured in LB medium and were further grown to an optical density at 600 nm (OD600 ) of 1.0. Total RNA from each strain was extracted using an RNAprep Pure Cell/Bacteria Kit with on-column DNase I digestion (TIANGEN Biotech, Beijing, China). RNA was further treated by DNase I and was purified using an RNAclean Kit (TIANGEN Biotech). RNA concentration and purity were evaluated with a NanoDropTM 20 0 0 spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was verified by denaturing agarose gel electrophoresis. cDNA was synthesised using a Super RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time reverse transcription PCR (RT-qPCR) was carried out using AceQ® qPCR SYBR® Green Master Mix (Vazyme Biotech, Nanjing, China). The 30S rRNA rpsL gene was used as an internal control to normalise gene expression quantities between samples. Primer sequences are listed in Supplementary Text S1.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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>1024 512 256 >128 >128 64 128 32 >128 >128 4 64 >256 16 256 >1024 128 64 >128 >128 32 32 16 >128 128 2 32 256 16 256 2 2 2 1 <0.125 <0.125 <0.25 <0.125 <0.125 <0.125 <0.125 <0.125 <0.125 <8 <8
[pUCP24::4blaGES-5 ] [pUCP24::blaGES-5 ]
1024 2 2 >128 8 4 1 1 2 2 1 4 1 32 <8
PA1280
>1024 1024 256 >128 >128 >128 32 64 >128 >128 16 32 256 64 <8
agent
AMP PIP TZP FOX CRO CTX CAZ FEP CFP CSL ATM IPM MEM KAN GEN
EC600 [pICP-4GES]
PA1280 MIC (μg/mL)
SMRT sequencing technology was applied to determine the complete nucleotide sequence of the conjugative plasmid. In addition, an Illumina genomic library was constructed and sequenced and was subsequently used to correct the preliminary assembly generated by the SMRT long reads. The results showed that P. aeruginosa PA1280 harboured a 50 914-bp circular plasmid containing a set of genes responsible for plasmid replication, partition, stable inheritance and conjugal transfer (Fig. 1A). The replication region (ssb and trfA) of the plasmid shared >99% identity with
Antimicrobial
3.2. Plasmid sequencing and comparative genome analysis
Table 1 Antimicrobial susceptibility testing of 12 strains.
Recently, we isolated a P. aeruginosa clinical strain, designated as PA1280, that was resistant to multiple β -lactam antibiotics, including carbapenems, and possessed particularly strong resistance to meropenem compared with commonly isolated carbapenemresistant strains. MLST showed that PA1280 belonged to ST1119. Plasmid extraction and agarose gel electrophoresis exhibited positive results. To address the concerns of acquired carbapenem resistance, the plasmid was eliminated through high-voltage electroporation, which significantly reduced the resistance activity against the selected β -lactams except for cefoxitin (Table 1). Conjugal transfer of the plasmid to E. coli EC600 enhanced the resistance of the transconjugants to a series of β -lactams, including piperacillin, cefoxitin and ampicillin, but not carbapenems, which is consistent with the observations from the resistance profiles of IBC-2 in E. coli [35], and the transconjugants’ susceptibility to carbapenem can be ascribed to the distinct mechanisms of antimicrobial resistance between P. aeruginosa and E.coli. These results indicated that the plasmid from P. aeruginosa PA1280 was conjugative and carried strong resistance determinants against carbapenems, which prompted us to determine its complete sequence.
EC600
3.1. Identification of a conjugative plasmid
512 16 8 32 0.5 <0.125 2 <0.125 <0.125 <0.125 <0.125 0.25 <0.125 <8 <8
PA1280 PA1280
3. Results and discussion
3 MIC, minimum inhibitory concentration; AMP, ampicillin; PIP, piperacillin; TZP, piperacillin/tazobactam; FOX, cefoxitin; CRO, ceftriaxone; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CFP, cefoperazone; CSL, cefoperazone/sulbactam; ATM, aztreonam; IPM, imipenem; MEM, meropenem; KAN, kanamycin; GEN, gentamicin.
512 32 8 64 16 16 4 4 32 16 4 1 16 64 64 128 8 4 64 8 8 2 2 16 4 2 0.5 4 64 64 >1024 512 256 >128 >128 64 32 32 >128 >128 8 4 >256 64 256 32 4 4 128 4 8 1 2 4 2 4 0.25 1 32 <8
>1024 256 64 >128 128 32 32 16 >128 128 4 2 128 64 256
PAO1PAO1-
blaGES-5 [pUCP24::4blaGES-5 ]
PAO1 PAO1 PAO1
[pUCP24::blaGES-5 ]
A class 1 integron with the gene arrangement intI1–blaCARB-2 – aadA2–cmlA1–aadA1 was stored in our laboratory and was used as template for PCR amplification. The fragment intI1–blaCARB-2 , which contains the intact intI1 and truncated blaCARB-2 (61bp coding sequence), was amplified and was cloned into the pUCP24 vector by EcoRI–BamHI digestion. The recombinant vector (pUCP24::intI1–blaCARB-2 , also named as Construct I) was transformed into EC600[pICP-4GES] by electroporation (25 μFD, 200 and 2.5 kV) and was selected on a compound LB agar plate containing 20 μg/mL gentamicin. Transformants were subjected to overnight culture and total plasmid extraction and were further chemically transformed into the E. coli DH5α recipient. Transformants that only harboured the blaGES-5 cassette-captured recombinant vector were screened using 100 μg/mL ampicillin, whereas transformants only harbouring Construct I without a cassette insertion were filtered. The original pICP-4GES could not be efficiently transformed into the E. coli DH5α recipient owing to its larger size. The same experimental procedure was also performed using the original pUCP24 for cassette capture. PCR and Sanger sequencing were used to validate the recombinant vector with the cassette insertion. The recombination frequency from Construct I to Recombinants I (blaGES-5 cassette integrated into attI1-I) and II (blaGES-5 cassette integrated into attI1-II) was calculated as the CFU/mL of the transformants selected by 100 μg/mL ampicillin divided by the CFU/mL of the transformants selected by 20 μg/mL gentamicin. Statistical analysis of the recombination frequency of two attI1 sites was performed by using two independent samples t-test.
4blaGES-5
P. aeruginosa ATCC 27853
2.7. Cassette capture assay
1024 2 2 >128 8 4 1 2 4 2 4 16 <0.125 512 16
T. Xu et al. / International Journal of Antimicrobial Agents 000 (2018) 1–7
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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Fig. 1. Genomic structure and comparative analysis of plasmid pICP-4GES from Pseudomonas aeruginosa PA1280. (A) Genomic structure of the conjugative plasmid pICP-4GES. Genes are denoted by arrows and are coloured based on the gene function classification. IRL and IRR represent left and right inverted repeats, respectively. (B) Comparative genome analysis of pICP-4GES with three other IncP-1β group plasmids. Orthologous genes are connected by lines and colour coded, except genes marked with grey colour. Genes labelled with hp represented hypothetical genes.
those well-documented plasmids belonging to the IncP-1β incompatibility group with broad host range, including R751, pB8 and pA22732-IMP [36–38]. Comparative genome analysis showed that the plasmid isolated from P. aeruginosa PA1280 exhibited highly global genomic synteny and possessed a backbone similar to the aforementioned IncP-1β plasmids, whilst the gene content and organisation in the two accessory regions were variable (Fig. 1B). One accessory region of the plasmid was horizontally transferred by a typical Tn3 family transposon identified by a pair of imperfect 35-bp inverted repeated (IRs) immediately flanking the region encoding from tnpA to lysR (Fig. 1A). Unexpectedly, the other accessory region included a class 1 integron harbouring four consecutive blaGES-5 gene cassettes. This class 1 integron contained the complete 5 -conserved segment (CS) but lacked the entire 3 -CS; the truncated trbN gene only encoded the first 30 amino acids of TrbN. Consideration of the above properties, this plasmid was named pICP-4GES. In addition, the coding orientation of the class 1 integron from pICP-4GES was different from pB8 and R751 but was the same as that of pA22732-IMP. Interestingly, compared with R751, pICP-4GES lacked the entire tni region at one end of the class 1 integron, and the trbO and trbP genes were also absent in contrast to the three other plasmids, suggesting that these three genes are not required for conjugation. Multiple identical gene cassettes captured by IntI1 appear to emerge rarely, although as many as eight gene cassettes have been found in a single class 1 integron [24]. To validate this genomic architecture, we first used a pair of blaGES-5 outward primers against P. aeruginosa PA1280 for PCR amplification. In addition to a bright band, there were obscure bands corresponding to the estimated sizes of amplicons spanning two (cassette 1–3 or 2–4) and three (cassette 1–4) cassettes in P. aeruginosa PA1280 (Supplementary Fig. S1). Similar results were observed using a pair of primers for amplification of the blaGES-5 complete ORF (Supplementary Fig. S1). Second, we realigned the Illumina reads to pICP-4GES. The results showed that the average sequencing depth at a one base resolution of the tandem blaGES-5 cassette region was 2684.14, which was ca. 4.00-fold greater than that of the remaining region (670.65) of the plasmid. Third, PCR amplification from the intI1 internal region to the region immediately downstream of the last cassette showed the expected product size, and Sanger sequencing from both ends extended into the blaGES-5 cassette, which supported the SMRT reads assembly. Thus, pICP-4GES harboured a class 1 integron with four copies of the blaGES-5 gene cassette.
3.3. Cassette stability Compared with other IncP-1β group plasmids, the terminal region of this class 1 integron appeared atypical. To evaluate the stability of four identical cassettes after the plasmid was transferred to the recipient, 20 rounds of culture of the transconjugant were performed in the absence of any antibiotics. A total of 100 randomly selected clones were used as template for PCR amplification of the four complete cassettes. The product sizes generated from all the clones were completely equal to that of the wild-type strain, indicating that the cassettes in the recipient strain were stable without undergoing major cassette copy changes. 3.4. Resistance activity differences between quadruple and single blaGES-5 A promoter, termed Pc, is located within the intI1 of class 1 integrons and initiates gene cassette expression [39]. A series of Pc variants have been identified and their activities associated with gene cassette transcription and recombination have been characterised [40,41]. The Pc sequence of the class 1 integron from pICP-4GES was analysed and the results showed that the promoter of the cassette belonged to PcW (weak Pc) (Table 2). To characterise the activity of four consecutive blaGES-5 gene cassettes associated with resistance to antimicrobials, both single and four blaGES-5 cassettes, including PcW, were cloned in the multiple copy vector pUCP24 and were further transformed into two recipients, PA1280 and PAO1, the latter being a standard P. aeruginosa strain whose ampG has been knockedout [33]. Compared with wild-type P. aeruginosa PA1280, both pUCP24::blaGES-5 and pUCP24::4blaGES-5 could fully complement or even enhance the resistance activity of PA1280 to several β -lactams, including meropenem, imipenem and ceftazidime, but not to piperacillin, cefotaxime, cefepime or aztreonam. The incomplete complementation of the resistance to these antimicrobials may be ascribed to another β -lactamase gene, blaP, located on the accessory region of pICP-4GES in the wild-type strain (Fig. 1A). Furthermore, pUCP24::blaGES-5 and pUCP24::4blaGES-5 also significantly enhanced the MICs of all selected β -lactams compared with PAO1, except for aztreonam. The contributions to β -lactam resistance by pUCP24::4blaGES-5 were generally higher than those of pUCP24::blaGES-5 for at least one MIC dilution both in PA1280 and PAO1 strains.
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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Table 2 Variants of gene cassette promoters involved in three class 1 integrons. Integron
Promoter Pc
intI1–4blaGES-5 intI1–blaPSE-1 –intI1–aacC1
Pc variant
–35 region
–10 region
TGGACA TGGACA TTGACA
TAAGCT TAAGCT TAAACT
PcW PcW PcS
P2
P2 variant
–35 region
–10 region
TTGTTA TTGTTA TTGTTA
TACAGT TACAGT TACAGT
P2 P2 P2
PcW, weak Pc; PcS, strong Pc.
Fig. 2. Schematic representation of the blaGES-5 gene cassette capture assay. (A) Genes are denoted by coloured arrows. Red circle represents the circular cassette excised from the donor plasmid and subsequently fixed into the corresponding attI1 site. Small paired red arrows indicate the relative positions of the PCR primers used for the cassette capture validation, whereas red–white chimeric arrows represent the relative position of the sequencing primers used for validation. (B) The nucleotide sequences of each original and new generated attI1 and attC sites. Red dashed boxes indicate the attI1 sites that were integrated with the gene cassettes. (C) Recombination frequency between blaGES-5 gene cassette and two different attI1 sites.
Because of the multi-copy status of pUCP24 in the host cell, to monitor and compare the resistance activity of either four or one blaGES-5 in a single copy state, the expression of both 4blaGES-5 and blaGES-5 downstream of the glmS gene in the PAO1 chromosome was targeted using a mini-Tn7 system [34]. Compared with PAO1, chromosome-targeted expression both of 4blaGES-5 and blaGES-5 significantly enhanced the resistance against selected β -lactams. In addition, compared with PAO1-blaGES-5 , PAO14blaGES-5 possessed a one to two MIC dilution higher resistance capacity against all the selected β -lactams except for cefoxitin.
3.5. Gene expression analysis of quadruple and single blaGES-5 cassettes Since the resistance activity of four copies of blaGES-5 was one to two dilution MICs higher than the single blaGES-5 -harboring strain, we wanted to identify the gene expression differences between them. Gene expression analysis of four bacterial strains, including wild-type PA1280, PA1280, PA1280[pUCP24::blaGES-5 ] and PA1280[pUCP24::4blaGES-5 ] was carried out by RT-qPCR during bacterial exponential-growth phase. Compared with
Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002
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PA1280[pUCP24::blaGES-5 ],
PA1280[pUCP24::4blaGES-5 ] increased blaGES-5 expression by 2.07-fold, indicating that the gene expression difference of blaGES-5 between two complementary strains was not directly correlated with the variation in gene copy number. This may have been because the strength of gene expression declines as cassettes become more distant from the promoter [39] and because of the non-additive nature of the contribution to MIC of antimicrobials by multiple identical determinants. In addition, gene expression of blaGES-5 in PA1280[pUCP24::blaGES-5 ] was 2.13-fold higher than P. aeruginosa PA1280 wild-type strain, which could be primarily attributed to the higher copy number of pUCP24 in the bacterial cell. However, blaGES-5 expression in PA1280 was at an undetectable level.
that four copies of blaGES-5 enhanced expression by only approximately two-fold compared with the single-copy strain that was driven by a weak cassette promoter. Funding This work was financially supported by the National Natural Science Foundation of China [grant nos. 81501780, 31500109, 81401702 and 81501808]; the Natural Science Foundation of Zhejiang Province, China [grant no. LQ17H190 0 01]; the Nucleic Acids Based Molecular Diagnostics Innovative Discipline, Health and Family Planning Commission of Zhejiang Province; and start-up funds from the Wenzhou Medical University to TX [grant no. QTJ14002].
3.6. Cassette transfer frequency The blaGES-5 gene cassette is widely distributed in naturally occurring class 1 integrons. In order to characterise the potential transfer efficiency of this cassette, a multi-copy vector pUCP24 was used for potential cassette capture assay, which could provide more opportunities for cassette attachment. In pICP-4GES, only the same cassette emerged, which provided a naturally simple cassette donor for quantification of the blaGES-5 recombination frequency and was thus used as cassette donor plasmid. In addition, a DNA fragment containing intact intI1 together with a truncated blaCARB-2 gene (intI1–blaCARB-2 ) was cloned into the pUCP24 vector (Fig. 2). The modified pUCP24 therefore contains two attI1 sites for potential cassette capture (Fig. 2, Construct I). One is an inherent attI1 site (attI1-II) and the other (attI1-I) is derived from the exogenous fragment. If the vectors capture a cassette by either attI1 site after their transformation into the donor plasmidcontaining strain, the two novel junctions formed by the cassette integration can be amplified by specific PCR primer pairs, which could result in products with an enlarged size in response to the non-cassette-capture vector and would not produce an amplicon with the cassette-donor plasmid (pICP-4GES) alone. Sequence analysis indicated that the Pc variant in intI1–blaCARB-2 was a weak promoter that is identical to that in pICP-4GES, whereas Pc identified from intI1–aacC1 was a strong one (Table 2). The original pUCP24 was transformed into EC600[pICP-4GES] for potential cassette capture. There was no cassette inserted into the inherent attI1-II site of original pUCP24, which was reflected by the phenotypic failure to be selected by the corresponding antimicrobial. However, the pUCP24::intI1–blaCARB-2 (Construct I) was transformed into transconjugants and the re-transformants exhibited the ampicillin-resistant phenotype, indicating that cassettes were fixed into the attachment sites. PCR and sequencing of the selected clones showed that both of the attachment sites were integrated by the blaGES-5 gene cassette. In addition, no cassette-donor plasmid could be detected in any re-transformants. We also did not detect a multiple tandem cassette inserted in any of the two attI1 sites. The relatively global recombination frequency of the cassette capture is ca. 5.38 × 10–4 . The attI1-II × attC recombination (Recombinant II) frequency was 5.04 × 10–4 , which was ca. 15-fold higher than that of the attI1-I × attC recombination (3.45 × 10–5 , Recombinant I; P < 0.001) (Fig. 2). This recombination frequency was ca. 3-fold lower than that of aadA2 cassette (1.87 × 10–4 ) that has been demonstrated to be extensively distributed in naturally occurring class 1 integrons [42]. In conclusion, this study characterised an unusual class 1 integron harbouring four copies of blaGES-5 . The atypical termination at the 3 -CS of the integron did not appear to affect the stability of the copy number of cassettes. The resistance activity of the four blaGES-5 genes was generally stronger than a single determinant with an approximately one to two dilution MIC of the selected β -lactam antibiotics. Gene expression analysis also showed
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Please cite this article as: T. Xu et al., Characterisation of a class 1 integron associated with the formation of quadruple blaGES−5 cassettes from an IncP-1β group plasmid in P seudomonas aeruginosa, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.07.002