First Report of bla OXA-677 with Enhanced Meropenem-Hydrolyzing Ability in Pseudomonas aeruginosa in China.

PURPOSE: OXA-10-type class D β-lactamases have shown their evolutionary potential of enhancing carbapenem resistance. This study aimed to elucidate the role of OXA-10 variants in clinical isolated multidrug resistant (MDR) Pseudomonas aeruginosa and characterize the first appearance of OXA-677 in China.
METHODS: Six bla OXA-10-like-positive strains were screened by PCR from 41 P. aeruginosa strains, which were resistant to both carbapenems and ceftazidime-avibactam, collected across China in 2018. The minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The resistance-associated genes and genetic environment were investigated by whole-genome sequencing (WGS). The function and mechanism of OXA-677 β-lactamase were identified by molecular cloning and protein structure modeling.
RESULTS: All the bla OXA-10-like-positive Pseudomonas aeruginosa were MDR strains. They also had outer membrane porin defects and produced β-lactam resistance gene bla PER-1, fluoroquinolone-resistant gene crpP, aminoglycoside-resistance gene aph(3')-IIb, aph(6)-Id, aacA and aadA, fosfomycin-resistance gene fosA, sulfamethoxazole-resistance gene sul1, and chloramphenicol-resistance gene catB7. All bla OXA-10 variants were located in a Tn1403-related transposon, containing aacA4-12-bla OXA-677-aadA1, aacA4-12-bla OXA-101-aadA5, and bla OXA-246-aacA3-aadA13 gene cassette arrays, respectively. Notably, the bla OXA-677 producer showed a high MIC level of meropenem (MIC>64 mg/L). Compared to bla OXA-10, bla OXA-677 was found a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117, which is closer to leucine155 in the omega loop of the active site. MIC of meropenem for E. coli DH5α with the recombinant plasmid pHSG398 carrying bla OXA-677 was elevated by 8 times.
CONCLUSION: We speculate that the OXA-10-like enzymes and the decrease of membrane permeability confer carbapenem resistance, and the V117 substitution in OXA-677 might lead to a higher resistance level of meropenem.

Introduction

Pseudomonas aeruginosa is one of the most important and common clinical opportunistic pathogens, and its infection carries a high risk of mortality.1 Due to various inherent and acquired resistance mechanisms, the multidrug-resistant P. aeruginosa has emerged globally.2,3 There into, carbapenem-resistant P. aeruginosa was included in the highest category of urgency by the World Health Organization in 2017.4 According to the China Antimicrobial Surveillance Network (CHINET) of 2020, the resistance rates of P. aeruginosa to imipenem and meropenem are 23.2% and 19.3%, respectively.5

One or more mechanisms, including alteration of outer membranes, efflux pump overexpression, and production of carbapenemases, have been found contributing to the carbapenem resistance in P. aeruginosa isolates.6–8 It is generally considered that only enzymes with broad-spectrum and significant carbapenem hydrolysis activities are the most predominant carbapenemases, such as the class B metal-β-lactamases NDM, IMP, VIM, and the class A serine-β-lactamases KPC. In recent years, special attention has been given to the class D serine-β-lactamase OXA-family.

The OXA-family counts 926 members and only several groups are considered as carbapenem-hydrolyzing class D β-lactamases (CHDLs), such as OXA-23-like, OXA-40/24-like, OXA-58-like, and OXA-48-like β-lactamases. Among these carbapenemases, only OXA-40-like genes have been identified in P. aeruginosa, and the rest are prevalent in A. baumannii. However, some studies suggest that OXA-10 and OXA-2 β-lactamases can also confer reduced susceptibility to carbapenems when they express in strains with outer membrane porin defects, just like the well-known CHDL OXA-48.9 Furthermore, modification of OXA-10 may enhance resistance to either carbapenem or new β-lactam/β-lactamase inhibitor combination.10,11 According to the NCBI database, a total of 37 OXA-10-like enzymes have been reported around the world so far, and over 75% (28/27) of them have been detected in P. aeruginosa.12–14 OXA-14, OXA-794, OXA-795, and OXA-824, confer resistance to β-lactam/β-lactamase inhibitor but decrease meropenem MICs.10 Kotsakis et al reported the first natural variant OXA-655 (V117L) with increased carbapenem hydrolysis produced by an environmental E. coli strain.15 In this study, a variant similar to OXA-655 (OXA-677, V117F) and other two variants (OXA-101 and OXA-246) were found to be produced by several clinically relevant carbapenem resistant P. aeruginosa isolates. Here, we tried to characterize the rare variant OXA-677 and elucidate the role of OXA-10 variants in carbapenem resistance.

Materials and Methods

Strains and Plasmid

P. aeruginosa strains named 18-W18-009, 18-W18-012, 18-W71-012, 18-W71-013, 18-W71-020, 18-W31-087 were found producing OXA-10-like enzymes during the screen for clinical isolates, which were resistant to both carbapenems and ceftazidime-avibactam among 39 strains by the CHINET workgroup in 2018. All strains were identified using VITEK matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, bioMérieux, Marcyl’Étoile, France). P. aeruginosa ATCC 27853 was used as the quality control strain for antimicrobial susceptibility testing; P. aeruginosa PAO1 was used as the reference strain for the whole-genome sequence blasting in the Genebank; E. coli DH5α and plasmid pHSG398 were used as the recipient bacterium for transformation test and the resistance gene carrier for clone experiment, respectively.

Antimicrobial Susceptibility Testing

Minimal inhibitory concentrations (MICs) were determined with broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines of the 30th edition. Antimicrobial agents used in antimicrobial susceptibility tests were imipenem, meropenem, ceftazidime/avibactam, piperacillin/tazobactam, ceftolozane/tazobactam, cefepime, amikacin, polymyxin B, ceftazidime, aztreonam, and ciprofloxacin. MIC interpretation for most agents was performed based on the CLSI breakpoints except for polymyxin, which was interpreted using EUCAST standards.16

Whole Genome Sequencing and Bioinformatics Analyses

Total genomic DNA was extracted using the genomic DNA extraction kit (Tiangen, China). Fragmented genomic DNA (~400-bp) was obtained through the Covaris S2 (Covaris, USA) and library construction was performed by NEXTflex DNA Sequencing Kit compatible with the Biomek® FXp (Bio Scientific, USA). Genome-wide sequencing was performed using Illumina Solexa technology (Illumina, San Diego, CA, USA). De novo assembly was carried out with Velvet v1.2.08 and redundant contigs were removed using the Fastx toolkit.

The multilocus sequence typing (MLST) was identified by an online Pseudomonas aeruginosa mlst typing database (). The presence of horizontally acquired resistance mechanisms was detected with the ResFinder v4.1 server () and the whole bacterial genome annotation was performed by Rapid Annotation using Subsystem Technology (RAST) (). The mobile element-related genes (MGEs) were identified by MobileElementFinder (), ISfinder (), and Tn Number Registry (). Additionally, gene alterations and disruptions of porin-encoding genes oprD and efflux pump repressor gene mexR, nalC, nalD were compared with those of P. aeruginosa PAO1 using SnapGene.

Molecular Cloning, Transformation Test and Protein Structure Modeling of blaOXA-677

The gene of blaOXA-677 was amplified with Q5® High-Fidelity 2X Master Mix. OXA-10-F-EcoRI (5ʹ-CGGAATTCGCCATGAAAACATTTGCCGCATATG-3ʹ) and OXA-10-R-BamHI (5ʹ -CGCGGATCCGTTAGCCACCAATGATGCCC-3ʹ) were used as the primer pair. The cycling parameters were 98°C for 30 s, 30 cycles at 98°C for 30 s, 63°C for 30 s and 72°C for 60 s. The PCR product was ligated to the vector plasmid pHSG398 and then the recombinant plasmid was transferred into the recipient strain E. coli DH5α. Mueller-Hinton agar plates containing 50 mg/L of chloramphenicol (for pHSG398) and 50 mg/L of amoxicillin (for blaOXA-677) were used to select the potential transformants, which were confirmed finally by PCR and sanger sequencing. The three-dimensional protein structure models for OXA-677 carbapenemase were built by SWISS-MODEL Server () and PyMOL software (), using that of OXA-655 carbapenemase (PDB accession number 6SKQ; resolution, 2.10Å) as a reference.

Nucleotide Accession Numbers

The sequences of OXA-10 variants and their gene context have been deposited in the GenBank database under accession numbers OL353898 (blaOXA-101), OL353899 (blaOXA-246) and OL353897 (blaOXA-677).

Results

Resistance Phenotypes and Genotypes of blaOXA-10 Variants Positive Strains

P. aeruginosa strains named 18-W18-009, 18-W18-012, 18-W71-012, 18-W71-013, 18-W71-020, 18-W31-087 expressed multidrug resistant phenotypes. They were all resistant to imipenem, meropenem, ceftazidime/avibactam, piperacillin/tazobactam, ceftolozane/tazobactam, cefepime, amikacin, ceftazidime, aztreonam, and ciprofloxacin. P. aeruginosa named 18-W31-087 showed a higher MIC level of meropenem (MIC > 64 mg/L) than others (MIC = 8–32 mg/L). No carbapenemase gene could be detected except blaOXA-10-like in these strains by the PCR (all carbapenemase genes tested in this study were shown in with primers sequence), as well as the following whole-genome sequencing (WGS). PCR and WGS data revealed that strain 18-W31-087 produced OXA-677 enzyme, strain 18-W18-009 and 18-W18-012 produced OXA-256 enzyme, strain 18-W71-012, 18-W71-013, 18-W71-020 produced OXA-101 enzyme. blaOXA-246-positive strains belonged to ST298, and others belonged to ST244. Meanwhile, chromosomal mutations such as premature termination of the efflux pump repressor gene mexR and porin-encoding gene oprD were observed in blaOXA-246-positive strains. And for strains producing OXA-677 or OXA-101 enzymes, premature stop codon occurred in oprD genes. Besides the carbapenem resistance associated genes, all the six strains produced extended spectrum β-lactamase (ESBL) gene blaPER-1, fluoroquinolone-resistant gene crpP, aminoglycoside-resistance gene aph(3ʹ)-IIb, aph(6)-Id, aacA and aadA, fosfomycin-resistance gene fosA, sulfamethoxazole-resistance gene sul1, and chloramphenicol-resistance gene catB7. blaOXA-246-positive strains also produced blaTEM-1B and aminoglycoside-resistance gene rmtB (Table 1).

Table 1

Demographic, Antimicrobial Susceptibility and Genotypic Data for the Clinical Isolates of P. aeruginosa Harboring OXA-10 Natural Variants

Strain ID	Hospital Location	Sample Type	MIC (mg/L)											MLST	β-Lactam Resistance Genes and OMP Mutation Sites	Non-β-Lactam Resistance Related Genotypes
			IPM	MEM	CZT	CZA	TZP	FEP	AMK	POL	CAZ	ATM	CIP
18-W18-009	Liaoning	Sputum	8	16	128	64	256	64	>128	>16	>32	>128	>8	ST298	OXA-246 (K138N), PER-1, TEM-1B, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA3, aadA13, rmtB, catB7
18-W18-012	Liaoning	Sputum	16	8	128	64	256	64	>128	1	>32	>128	>8	ST298	OXA-246 (K138N), PER-1, TEM-1B, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA3, aadA13, rmtB, catB7
18-W71-012	Inner Mongolia	Urine	32	16	128	16	>256	64	128	0.5	>32	>128	>8	ST244	OXA-101 (I10T G20S S27F D55N T107S Y74F), PER-1, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA4-12, aadA5, catB7
18-W71-013	Inner Mongolia	Sputum	32	32	>128	32	>256	128	>128	0.5	>32	>128	>8	ST244	OXA-101 (I10T G20S S27F D55N T107S Y74F), PER-1, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA4-12, aadA5, catB7
18-W71-020	Inner Mongolia	Sputum	32	32	>128	32	>256	128	128	1	>32	>128	>8	ST244	OXA-101 (I10T G20S S27F D55N T107S Y74F), PER-1, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA4-12, aadA5, catB7
18-W31-087	Shandong	Secretion	32	>64	>128	32	>256	>128	>128	2	>32	>128	>8	ST244	OXA-677 (V117F), PER-1, OprD (237stop)*	crpP, fosA, sul1, aph(3ʹ)-IIb, aph(6)-Id, aacA4-12, aadA1, catB7

Note: *Frameshift caused by base-deletion.

Abbreviations: IPM, imipenem; MEM, meropenem; CZA, ceftazidime/avibactam; TZP, piperacillin/tazobactam; FEP, cefepime; AMK, amikacin; POL, polymyxin B; CAZ, ceftazidime; ATM, aztreonam; CIP, ciprofloxacin; OMP, outer membrane proteins.

Genetic Context and Protein Structure of blaOXA-10 Variants

All three kinds of blaOXA-10 variants are located in a Tn1403-related transposon of the Tn-3 family. Each transposon contains a complex class 1 integron harboring an ISCR1-blaPER-1 unit. The difference of the three class 1 integrons was gene cassette arrays (GCA) in the variable region (VR). GCA aacA4-12-blaOXA-677-aadA1, aacA4-12-blaOXA-101-aadA5, and blaOXA-246-aacA3-aadA13 were detected, respectively (Figure 1). Compared to the blaOXA-10, the sequence of blaOXA-677 was found carrying a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117 (Val117Phe); whereas there are no base mutations for either blaOXA-101 or blaOXA-246 at the key amino acid motifs (Figure 2). Val117Phe (V117F) was located at the second catalytically important conserved motif of class D β-lactamases. Due to the substitution, the phenylalanine117 is closer to leucine155 in the omega loop (Figure 3).

Figure 1

The genomic context of the blaOXA-677 (A), blaOXA-101 (B), blaOXA-246 (C). Genes are denoted by arrows. Genes, MGEs, and other features are colored based on their functional classification.

Figure 2

Amino acid alignment of OXA-10 and three OXA-10-like enzymes identified in this study. A dash indicates synonymous substitution without amino acid change. Key amino acid motifs conserved among OXA-10 are highlighted in red.

Figure 3

Superposition of OXA-10/OXA-101/ OXA-246 (blue), OXA-655 (yellow), and OXA-677 (red) with detailed view of active site. Leu155 and the omega loop are shown and labeled by the three mixed colors. Meropenem is depicted in green as a substrate. In the OXA-677 enzyme, the phenylalanine117 is closer to leucine155 in the omega loop.

Characteristic of Transformants Carrying blaOXA-677

Transformants were obtained from the screening plates after verification of an 822bp PCR-amplified product of blaOXA-677. Two sequence-confirmed transformants were randomly selected for antimicrobial susceptibility tests to ensure consistent results. Compared with the empty host, MIC of meropenem for the E. coli DH5α transformant with the recombinant plasmid pHSG398 carrying blaOXA-677 was elevated by 8 times (Table 2). No significant increase was observed for imipenem, as well as ceftolozane/tazobactam, piperacillin/tazobactam, ceftazidime/avibactam, ceftazidime, aztreonam, cefepime, amikacin, polymyxin B, and ciprofloxacin (Table 2).

Table 2

β-Lactam Resistance Profile Conferred by OXA-677 in E. coli DH5α Under Isogenic Conditions

Antimicrobial Agents	MIC (mg/L)				Ration of A/B
	Donor Strain	(A) DH5α-pHSG398-blaOXA-677	DH5α-pHSG398	(B) Recipient Strain (DH5α)
Imipenem	32	0.25	0.125	0.125	2
Meropenem	>64	0.125	0.015	0.015	8
Ceftolozane/tazobactam	>128	0.25	0.5	0.25	1
Piperacillin/tazobactam	>256	8	4	4	2
Ceftazidime/avibactam	32	0.125	0.125	0.06	2
Ceftazidime	>32	0.5	0.5	≤0.5	/
Ceftriaxone	>32	≤32	≤32	≤32	/
Cefuroxime	>32	8	16	8	1
Aztreonam	>128	≤1	≤1	≤1	/
Cefepime	>128	0.125	0.125	0.06	2
Amikacin	>128	≤1	≤1	≤1	/
Polymyxin B	2	0.25	0.25	0.25	1
Ciprofloxacin	>8	≤8	≤8	≤8	/

Discussion

OXA-10-like enzymes are widespread in P. aeruginosa and A.baumannii, and less common in Enterobacterales in Asia, Europe, South America, and Africa. It is the first report of the existence of blaOXA-10-like in ST298 and ST244 clones in China, which are two of the top 10 high-risk MDR/XDR P. aeruginosa clones causing nosocomial infection worldwide.17 blaOXA-101 was first reported in Enterobacteriaceae, Citrobacter freundii, Escherichia coli, and Enterobacter cloacae in Argentina in 2005 and now has been detected in A. baumannii and P. aeruginosa in China.18–20 blaOXA-246 was first found among ceftazidime-resistant P. aeruginosa clinical isolates in China in 2007.21 blaOXA-677 and its GCA aacA4-12-blaOXA-677-aadA1 were found for the first time in China in this study. Our study showed that these three variants were all embedded in class 1 integrons harboring an ISCR1-blaPER-1 unit. In public databases, the most similar integron was In1079, located on the chromosome of P. aeruginosa.22

Previous studies showed that the lack of OprD protein could only raise meropenem to 2–4 mg/L.23 Antunes et al suggested that the OXA-10 β-lactamase, which is regarded as the narrow-spectrum enzyme, is CHDL in fact.9 We speculate that the production of OXA-10-like enzymes and decrease of membrane permeability may cooperate kindly to confer meropenem resistance, for we found premature termination of oprD genes for all six blaOXA-10 variants positive strains in this study. Moreover, the antimicrobial susceptibility data of the clinical isolates and transformants showed that blaOXA-677 producing strain had a higher MIC level of meropenem. However, there was no difference between the resistance genes of blaOXA-677-positive strain and blaOXA-101-positive strains other than blaOXA-10-like genes. Structural bases of OXA-677 may explain the high-level resistance. As previously shown, OXA-677 has a single mutation at Val117. The activity of class D β-lactamases, including OXA-10, is dependent on Lys70 carboxylation in the active site. Val117 of the S115A116V117 catalytic motif is the component of the hydrophobic core surrounding Lys70 and is strictly conserve.24,25 It was validated in OXA-655 with V117L that alterations of the interactions with carbapenems were brought by substitutions at this key site.26 In both crystal structures of OXA-655 and OXA-677 proteins, their position 117 residues are closer to Leu155 in the omega loop, and their interactions seem stronger. This conformational change probably alters the accessibility of the meropenem to the active site. Further work is required to demonstrate the correlation between its structure and function.

In addition, all OXA-10 variants-producing isolates in this study harbor ISCR1-blaPER-1. Recent studies have shown that blaPER-1 may be a source of resistance to the new class of β-lactam/β-lactamase inhibitors ceftazidime/avibactam and ceftolozane/tazobactam.27 Therefore, these β-lactamase genes combined with porin loss may confer resistance to almost all kinds of carbapenem and β-lactam/β-lactamase inhibitors. Coupled with other gene cassettes (aadA, aacA, sul1) in Tn3-like transposons conferring resistance to fluoroquinolone, aminoglycosides, and sulfonamides, these clinical isolates may be more likely to develop to MDR/XDR strains. And these gene cassettes might be transmitted among different strains due to the mobile genetic element. It is unfortunate from a clinical treatment standpoint for there might be no antibiotic available for these strains. Polymyxin B might be the only treatment option, albeit with toxicity. Therefore, screening for OXA-10 mutants during the treatment of P. aeruginosa infection may have some instructive significance for adjustment for medication use.

Conclusion

In summary, globally widespread OXA-10-like enzymes have shown their evolutionary potential toward enhancing carbapenem resistance. In this study, OXA-677 with increased carbapenemase activity was identified in a clinical MDR P. aeruginosa strain for the first time in China. It is also the first report of the existence of OXA-10 variants (OXA-677, OXA-101, and OXA-246) in ST298 and ST244 clones in China. Our findings provide evidence supporting the claim that OXA-10 should be classified as CHDL. It may help elucidate the role of OXA-10 variants in MDR P. aeruginosa strains. blaOXA-10-like along with other gene cassettes in class 1 integrons and membrane permeability defects contribute to the resistance to almost all available antibiotics, which brings challenges in their treatment. Further work with a larger number of positive strains is now needed to elucidate the mechanism of OXA-677 and other OXA-10 mutants.