Emergence of an Extensive Drug Resistant Pseudomonas aeruginosa Strain of Chicken Origin Carrying bla_IMP-45, tet(X6), and tmexCD3-toprJ3 on an Inc_pRBL16 Plasmid

ABSTRACT

This study reports an extensively drug resistant Pseudomonas aeruginosa strain PA166-2 which was of chicken origin and carrying bla_IMP-45, tet(X6) and tmexCD3-toprJ3 on a single plasmid. The strain was characterized by antimicrobial susceptibility testing, resistance gene screening, conjugation assay, whole-genome sequencing, and bioinformatics analysis. Strain PA166-2 was resistant to tigecycline and carbapenems. It belonged to ST313 and carried a plasmid pPA166-2-MDR, which belongs to the incompatibility group Inc_pRBL16. pPA166-2-MDR harbored a 78 Kb multidrug resistance (MDR) region carrying an array of antimicrobial resistance genes, including bla_IMP-45, tet(X6), and tmexCD3-toprJ3. The gene bla_IMP-45 was inserted into the backbone of plasmid pPA166-2-MDR within a class 1 integron, In786. tmexCD3-toprJ3 in plasmid pPA166-2-MDR was inserted in umuC, constituting the genetic context of ISCfr1-tnfxB3-tmexC3-tmexD3-toprJ3-△umuC. The genetic context of tet(X6) in this plasmid was identical to that of other reported plasmid-borne tet(X) variants, namely, tet(X6)-abh-guaA-ISVsa3. To the best of our knowledge, this is the first report of the cooccurrence of bla_IMP-45, tet(X6), and tmexCD3-toprJ3 in one plasmid in Pseudomonas sp. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes from chickens expanded the global transmission of vital resistance genes. Findings from us and from others indicate that plasmids of the incompatibility group Inc_pRBL16 may serve as a reservoir for carbapenem and tigecycline resistance determinants.

IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes infections that are difficult to treat. This study reported, for the first time, the occurrence of last-resort antibiotic resistance determinants bla_IMP-45, tet(X6), and tmexCD3-toprJ3 on a single plasmid in P. aeruginosa from chickens. The P. aeruginosa strain belonged to ST313 and was resistant to last-line antibiotics, namely, carbapenems and tigecycline. The plasmid carrying the last-line resistance genes belonged to the incompatibility group Inc_pRBL16, which was reported to contain different profiles of accessory modules and thus carried diverse collections of resistance genes. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes, from chickens expanded the global transmission of vital resistance genes. The results in this study highlighted that Inc_pRBL16 plasmids may serve as a reservoir for the dissemination of resistance genes. Control measures should be implemented to prevent the further dissemination of such strains.

OBSERVATION

Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals (1). The treatment of P. aeruginosa infections has become a significant challenge due to its remarkable capacity to resist many of the currently available antibiotics (2). P. aeruginosa exploits intrinsic, acquired, and adaptive resistance mechanisms to counter antibiotic attacks (3). Efflux pumps belonging to the plasmid-mediated resistance–nodulation–division (RND) family play a prominent role in the multidrug resistance of P. aeruginosa. Recently, a novel RND-type efflux pump gene cluster, tmexCD1-toprJ1, and its homologs, tmexCD2-toprJ2 and tmexCD3-toprJ3, were reported to confer resistance to different classes of antibiotics, including the last-line antibiotic, tigecycline (4–7). The tmexCD-toprJ gene clusters were speculated to have originated from the chromosome of a Pseudomonas species and disseminated among diverse bacterial species, including Pseudomonas sp., Klebsiella sp., Aeromonas sp., Enterobacter sp., Proteus sp., and Raoultella sp. (4–8). The coexistence of tmexCD-toprJ with other antimicrobial resistance genes, such as the colistin resistance gene mcr, the high-level mobile tigecycline resistance gene tet(X), and the carbapenemase genes bla_NDM and bla_KPC in single isolates, has been reported, particularly in Klebsiella sp. (5, 9, 10). The spread of mobile elements cobearing different last-line antimicrobial resistance determinants seriously compromises the effectiveness of clinical therapy. In this study, we report an XDR P. aeruginosa strain that co-harbors bla_IMP-45, tet(X6), and tmexCD3-toprJ3 on an Inc_pRBL16 plasmid of chicken origin. Heightened efforts are needed to control the dissemination of such strains.

P. aeruginosa strain PA166-2 was isolated from the cloaca swab of a chicken in a poultry farm in Shanxi, China in 2019. Antimicrobial susceptibility testing was conducted via the broth dilution method, and the results suggested that PA166-2 was resistant to tetracyclines (doxycycline and minocycline), a glycylcycline (tigecycline), carbapenems (meropenem and imipenem), some β-lactams (ceftazidime, cefepime, piperacillin-tazobactam, cefoperazone/sulbactam, ceftazidime-avibactam), ciprofloxacin, and an aminoglycoside (amikacin). The strain also exhibited intermediate resistance to colistin. However, the strain remained susceptible to aztreonam. The antimicrobial resistance profiles and mechanisms of resistance of P. aeruginosa PA166-2 are shown in Table 1. According to the nonsusceptibility level of strain PA166-2, it was classified as an extensive drug resistant (XDR) strain which was resistant to at least one agent in all but two or fewer antibiotic categories. Carbapenem resistance in P. aeruginosa is frequently associated with the expression of carbapenemase genes, so genes, including bla_IMP, bla_NDM, bla_VIM, bla_KPC, and bla_OXA, were screened via polymerase chain reaction (PCR) and Sanger sequencing, using primers described previously (11). A bla_IMP gene was detected positive. Meanwhile, strain PA166-2 was positive for the RND-type efflux pump gene cluster tmexCD-toprJ and for the tet(X) gene, both of which were recently reported to have conferred resistance to tigecycline (4, 12, 13). The antimicrobial resistance gene screening results were in line with the antimicrobial susceptibility testing (AST) results.

TABLE 1.

Results of antimicrobial susceptibility tests and genetic characterization^a

Antimicrobial agents	MIC (mg/L)	Interpretation	Mechanism of resistance/location of resistance gene
Aminoglycosides
Amikacin	≥128	R	aph(3′)-VIa, aph(3′)-Ic, aph(4)-Ia, armA, aac(3)-Iva, ant(3″)-Ih-aac(6′)-IId, /plasmid; aph(3′)-IIb/chromosome
β-Lactams
Imipenem	4	R	blaIMP-45/plasmid
Meropenem	32	R	blaIMP-45/plasmid
Ceftazidime	>128	R	blaIMP-45/plasmid
Cefepime	128	R	blaIMP-45 and blaOXA-1/plasmid
Piperacillin-tazobactam	128/4	R	blaIMP-45 and blaOXA-1/plasmid
Cefoperazone/sulbactam	>128/64	R	blaIMP-45/plasmid
Ceftazidime-avibactam	>64/4	R	blaIMP-45/plasmid
Aztreonam	≤4	S	-
Fluoroquinolones
Ciprofloxacin	16	R	qnrVC1 and tmexCD3-toprJ3/plasmid
Tetracyclines
Doxycycline	>32	R	Intrinsic resistance; tet(C), tet(X6) and tmexCD3-toprJ3/plasmid
Minocycline	32	R	Intrinsic resistance; tet(C), tet(X6) and tmexCD3-toprJ3/plasmid
Glycylcyclines
Tigecycline	16	R	Intrinsic resistance; tmexCD3-toprJ3 and tet(X6)/plasmid
Polymyxins
Colistin	1	I	-
Trimethoprim-sulfamethoxazole
Not included in the AST panel	NA	NA	sul1 and dfrA22e/plasmid
Macrolide, lincosamide and streptogramin B antibiotics
Not included in the AST panel	NA	NA	msr(E) and mph(E)/plasmid

^aR, resistant; S, susceptible; I, intermediate; NA, not applicable; -, none.

To decipher the genomic characterization, the genome of PA166-2 was extracted from overnight cultures by using the PureLink Genomic DNA Minikit (Invitrogen, Carlsbad, CA, USA) and sequenced by using the Illumina NextSeq 500 sequencing (2 × 150 bp) platform and the Nanopore MinION sequencer platform (14). The hybrid assembly of both sequencing reads was constructed using Unicycler v0.4.9b (15). The assembled genome of PA166-2 was annotated with the rapid antimicrobial susceptibility testing (RAST) tool and edited manually (16). Multilocus sequence typing was conducted by using the mlst software package (17). Antimicrobial resistance genes were analyzed by using ResFinder 2.1 (18). The genome of strain PA166-2 contained a 431,461 bp plasmid that was designated pPA166-2-MDR and a chromosome which was assembled into two contigs with lengths of 6,438,660 bp and 116,925 bp, respectively. The overall chromosome content of strain PA166-2 was comprised of 6,732 predicted open reading frames (ORFs), with a guanine-cytosine (GC) content of 65.6%. Antimicrobial resistance genes, including fosA, catB7, bla_OXA-50, aph(3′)-IIb, and bla_PAO, were detected on the chromosome of PA166-2. MLST analysis suggested that strain PA166-2 belonged to ST313. Plasmid pPA166-2-MDR contained 493 ORFs with a GC content of 56.3%. Two plasmids, pBM413 (CP016215) and pR31014-IMP (MF344571), both of which have similar backbones to that of pPA166-2-MDR, were retrieved from the NCBI nr database via a nucleotide Basic Local Alignment Search Tool (BLASTn) analysis (Fig. 1A). Plasmids belonging to the incompatibility group Inc_pRBL16 that contained diverse collections of resistance genes were recently reported in Pseudomonas spp. (19). Conserved Inc_pRBL16 backbone marker repA_IncpRBL16 together with its iterons, parB2-parA, che, pil, and ter, were detected on pPA166-2-MDR, pBM413, and pR31014-IMP, suggesting that they all belonged to the Inc_pRBL16 plasmid. An array of different resistance genes containing tmexCD3-toprJ3, bla_IMP-45, bla_OXA-1, tet(X6), tet(C), mph(E), msr(E), armA, sul1 (2 copies), catB3, qnrVC1, arr-3, floR, strAB (2 copies), ant(3′')-Ih-aac(6′)-IId, dfrA22e, aph(3′)-VIa, aph(4)-Ia, aac(3)-IVa, and aph(3′)-Ic were found in plasmid pPA166-2-MDR. Notably, this is the first known report of the cooccurrence of bla_IMP-45, tet(X6), and tmexCD3-toprJ3 in one plasmid. The multidrug resistance (MDR) region containing all of these acquired resistance genes was 78,304 bp in length and was similar to the corresponding region in pR31014-IMP, except for the presence of a ca. 19 Kb region harboring tet(X6) in pPA166-2-MDR. As in other Inc_pRBL16 plasmids, diverse mobile genetic elements, including TnAs1, intI1 (2 copies), ISCR1, ISEc28, IS1349, ISEc29, IS6100 (2 copies), ISEc59, Tn5393, ISVsa3, ISCfr1, and IS26 (4 copies) were detected in this MDR region (Figure 1B), suggesting that it was acquired via horizontal gene transfer and that active genetic recombination could have occurred in this region. A conjugation assay was performed via the filter mating method, using E. coli EC600 and fosfomycin-resistant P. aeruginosa PAO1 as the recipients. Transconjugants were selected on LB agar plates containing 1 mg/L meropenem and 600 mg/L rifampicin or 150 mg/L fosfomycin, respectively. Plasmid pPA166-2-MDR could not be transferred to P. aeruginosa and E. coli via direct conjugation under laboratory conditions.

FIG 1.

Detailed information regarding plasmid pPA166-2-MDR from Pseudomonas aeruginosa carrying an MDR region that encodes bla_IMP-45, tet(X6) and tmexCD3-toprJ3. (A) Comparative structural analysis of pPA166-2-MDR with other similar plasmids available in the NCBI nr database. The outermost circle represents the reference plasmid pPA166-2-MDR. The MDR region is highlighted with dotted lines. The acquired antimicrobial resistance genes, namely, conserved Inc_pRBL16 backbone markers repA_IncpRBL16 together with its iterons, parB2-parA, che, pil, and ter are annotated. (B) Alignment of the 78,304 bp MDR region in plasmid pPA166-2-MDR with the similar region in plasmid pR31014-IMP. Red, cyan, and orange arrows denote antimicrobial resistance genes, mobile elements, and other protein-encoding genes, respectively. Alignment of genetic contexts of tmexCD3-toprJ3 (C) and tet(X6) (D) in plasmid pPA166-2-MDR with similar sequences. Red, cyan, and orange arrows denote antimicrobial resistance genes, mobile elements, and other protein-encoding genes, respectively. The Δ symbol indicates that the gene is truncated.

tet(X6), which is a variant of the tet(X) gene that confers high-level tigecycline resistance, was first reported on an SXT/R391 element, ICEPgs6Chn1, in Proteus sp. (20). Previous studies have demonstrated that tet(X6) is frequently associated with the genetic context of tet(X6)-abh-guaA-ISVsa3, which is highly similar to that of other reported plasmid-borne tet(X) variants that are flanked by one or two ISVsa3 elements (20, 21). Likewise, pPA166-2-MDR carried tet(X6)-abh-guaA-ISVsa3 genetic content, and the ISVsa3 element upstream of tet(X6) was absent. Highly similar to that in ICEPgs6Chn1, the tet(X6) in pPA166-2-MDR was downstream of a truncated Tn5393 (Figure 1D). A BLASTn search in NCBI suggested that the tet(X) genes were absent on the Inc_pRBL16 plasmids in the database. tmexCD3-toprJ3 was also first reported in Proteus sp. on an SXT/R391 element, ICEPmiChnRGF134-1 (7). Previous studies have shown that most transposition units containing the tmexCD3-toprJ3-like gene clusters inserted into a similar site in the umuC gene (7, 22). In line with these findings, tmexCD3-toprJ3 in plasmid pPA166-2-MDR was found to be inserted in umuC, constituting the genetic context of ISCfr1-tnfxB3-tmexC3-tmexD3-toprJ3-△umuC. A BLASTn search with this genetic element in the NCBI nr database returned 8 hits (MF344570, KY883660, CP016215, CP086014, MF344568, CP073081, MN208062, and MF344571) with >98.5% identity at 100% coverage. All 8 of the sequences were from plasmids that belonged to the incompatibility group Inc_pRBL16 (Figure 1C). The bla_IMP-45 gene in pPA166-2-MDR was located directly downstream of the transposable element TnAs1 and in a class 1 integron, In786, with the gene arrangement intI1-aacA4-bla_IMP-45-bla_OXA-1-catB3. In786 was located within a Tn6485b transposon in pPA166-2-MDR. Similar genetic contexts were detected or reported in several other Inc_pRBL16 plasmids, including pBM413 and pR31014-IMP (Figure 1B) (19, 23, 24). Our findings suggested that Inc_pRBL16 was an important vector for the dissemination of last-line antibiotic resistance genes in Pseudomonas sp. The spread of plasmids like pPA166-2-MDR is of great concern for public health.

P. aeruginosa strains belonging to ST313 were widely disseminated across different continents (25). They have been described as intestinal colonizers in healthy individuals but were rarely reported from the poultry farm environment (26). The detection of such a strain in a chicken in this study suggested that this poultry could have been contaminated by human activities. Infections caused by P. aeruginosa are challenging to treat due to the intrinsic resistance of this bacterium to many antipseudomonal agents as well as its ability to acquire resistance determinants (2). ST313 P. aeruginosa were frequently reported to be associated with antimicrobial resistance genes, such as the carbapenemase genes bla_VIM and bla_KPC (27). However, the presence of Inc_pRBL16 MDR plasmids in ST313 P. aeruginosa was not reported previously. The acquisition of the Inc_pRBL16 plasmid carrying last-resort antimicrobial resistance genes bla_IMP-45, tet(X6), and tmexCD3-toprJ3 by P. aeruginosa pose considerable threats to public health.

In conclusion, this study reported, for the first time, the occurrence of last-resort antibiotic resistance determinants bla_IMP-45, tet(X6), and tmexCD3-toprJ3 on a single plasmid in P. aeruginosa from a chicken. The results of this study highlighted that Inc_pRBL16 plasmids may serve as a reservoir for the dissemination of resistance genes. Control measures, such as strict supervision, the application of laws to control antibiotic use, and timely screening, should be implemented to prevent the further dissemination of such strains.

Data availability.

The complete genome sequence of strain PA166-2 has been deposited in the NCBI GenBank database under the BioProject accession number PRJNA798590.