PFM-Like Enzymes Are a Novel Family of Subclass B2 Metallo-β-Lactamases from Pseudomonas synxantha Belonging to the Pseudomonas fluorescens Complex

TEXT

Metallo-β-lactamases (MBLs) are zinc-dependent enzymes that can catalyze the hydrolysis of virtually all β-lactam antibiotics (including carbapenems) except for monobactams and that are resistant to the β-lactamase inhibitors clavulanate, tazobactam, and avibactam (1). They constitute a highly diverse family of enzymes and can be categorized into three subclasses, namely, B1, B2, and B3 (2). The subclass B1 enzymes are the most clinically important since they comprise MBLs such as IMP-1, NDM-1, SPM-1, KHM-1, VIM-1, and VIM-2 (3), widely identified in Enterobacteriaceae, Acinetobacter spp., and Pseudomonas spp. Subclass B2 includes CphA (4, 5), ImiS (6, 7), and AsbM1 (8), which are intrinsic enzymes in Aeromonas spp., and Sfh-I (9) from the occasionally pathogenic species Serratia fonticola. These carbapenemases are monozinc enzymes that usually shown much higher hydrolysis rates against carbapenem substrates than the other β-lactams (9).

Production of MBLs in the Pseudomonas genus is frequently observed, with acquired MBL-encoding genes (bla_IMP, bla_VIM, bla_SPM) being reported worldwide mainly in Pseudomonas aeruginosa and, to a lesser extent, in Pseudomonas fluorescens (10, 11). In addition, intrinsic MBL genes encoding subclass B3 POM-1-like and PAM-1-like enzymes have been identified in Pseudomonas otitidis and Pseudomonas alcaligenes, respectively (12–14).

P. fluorescens and related species belonging to a same complex are rarely associated with infections in human medicine (15). Nevertheless, P. fluorescens can cause bloodstream infections in humans, and most reported cases have been iatrogenic (16). Few studies have focused on the β-lactamase gene content of the P. fluorescens complex. While P. fluorescens possesses a chromosomally located and inducible Ambler class C β-lactamase gene (17), the acquired but chromosomally located bla_BIC-1 gene encoding an Ambler class A carbapenemase was previously identified as a source of carbapenem resistance in P. fluorescens isolates recovered from the Seine River, Paris (18).

Here, we analyzed a carbapenem-resistant Pseudomonas sp. isolate that had been recovered during a survey aimed to study the spread of multidrug-resistant Gram-negative organisms among food varieties and food-producing animals in Switzerland in 2018. Isolate MCP-106 was isolated from chicken meat after an 18-h preenrichment in LB broth and subsequent selection on ChromID CarbaSmart (bioMérieux, La Balme-les-Grottes, France). Carbapenemase production was tested using the Rapid Carba NP test (19). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis assigned the strain to the Pseudomonas synxantha species, and that assignment was further confirmed by analysis of the rpoB and rpoD gene sequences (Fig. 1). P. synxantha, which belongs to the P. fluorescens complex (20), is an environmental species that reduces and accumulates the heavy metal chromium (21, 22) that is pathogenic to nematode eggs and may therefore be used as a nematicidal agent (23).

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FIG 1

Dendrogram performed by using the seven genes from the multilocus sequence typing (MLST) analysis in comparison with representative genes from other Pseudomonas species, in particular, the most closely related ones, which are Pseudomonas fluorescens and Pseudomonas synxantha. The alignment used for the tree calculation was performed with the Clustal Omega program.

Susceptibility testing performed for β-lactams by disk diffusion showed that P. synxantha strain MCP-106 was resistant to amino- and carboxypenicillins, broad-spectrum cephalosporins, aztreonam, and carbapenems. Whole-genome sequencing was performed using an Illumina MiSeq platform (2 × 150-bp paired ends) to assess the genetic determinants of carbapenem resistance. The obtained reads were trimmed using trimmomatic 0.36, assembled with SPAdes version 3.11.1 (24), and annotated with PROKKA version 1.12. TBLASTN analysis of the DNA contigs using VIM as a reference revealed a chromosomally located MBL protein that was named PFM-1 (Pseudomonas fluorescens metallo-β-lactamase). PFM-1 (encoded by the bla_PFM-1 gene) consisted of a β-lactamase with 253 amino acids and a relative molecular mass of 28.5 kDa.

A BLASTN analysis against the NCBI database revealed the presence of a bla_PFM-like gene (named bla_PFM-2, with PFM-2 sharing 92% amino acid identity with PFM-1) in Pseudomonas libanensis strain CIP105460 (GenBank accession no. GCA_001439685.1) (25) which actually belongs to the Pseudomonas fluorescens sp. complex. In addition, genes encoding PFM-like products were also identified in the genomes of a single P. fluorescens strain (WP_050516231.1) and two Pseudomonas brenneri strains, sharing 90% amino acid identity with PFM-1 (WP_128593843.1 and OAE14554.1). Furthermore, a gene encoding a more distantly related enzyme (75% amino acid identity) was found in the genome of a Pseudomonas chlororaphis strain (WP_038635452.1). However, no other bla_PFM-like gene was identified in any other P. fluorescens genomes (or in any genomes of species belonging to the same complex), despite numerous genomes of strains belonging to the P. fluorescens complex (n = 145) having been fully sequenced.

We then screened 10 P. fluorescens strains from our laboratory collection, all of which had been recovered from human, animal, or environmental samples. A PCR-based approach using primer pair PFM-1-Fw (5′-GTTACGCCTGATGGACTTTG-3′) and PFM-1-Rv (5′-CTTAGAAGCATGTCAGTGCG-3′) for bla_PFM-1 and primer pair PFM-2-Fw (5′-CTGATCAGAAAATGTGGGGC-3′) and PFM-2-Rw (5′-GACACGCCGTGTTTCTATATC-3′) for bla_PFM-2 was employed. A single strain gave a positive result, and Sanger sequencing identified a bla_PFM-like gene (bla_PFM-3) encoding a protein sharing 91% amino acid identity with PFM-1. The bla_PFM-3 gene was identified from P. fluorescens PF1, an isolate recovered from a water sample from the Seine River in Paris, France, and also producing the Ambler class A carbapenemase BIC-1 (18). PFM-2 and PFM-3 differed by five amino acids.

Pairwise alignment of the sequences of the PFM-like amino acid sequences with those of other MBLs revealed that these newly identified enzymes were most closely related to the subclass B2 MBL enzymes. PFM-1 shares 71% amino acid identity with Sfh-1, originally identified in Serratia fonticola strain UTAD54 (9), and 53% identity with CphA-1 from Aeromonas hydrophila (26). It shared very low identity with subclass B1 MBLs such as NDM-1 (17%) and VIM-1 and IMP-1 (22%) (Fig. 2). Protein alignments of the β-lactamase PFM-1 with representative subclass B2 MBLs revealed the presence of conserved amino acid residues known to be involved in binding to zinc of class B β-lactamase (BBL) (27) (Fig. 3). The motif Asn-Tyr-His-Thr-Asp (positions 116 to 120 [BBL nomenclature]), being a distinctive feature of subclass B2 MBLs and presumably involved in the coordination of the two zinc ions found in the active site of these enzymes, was identified in PFM-like enzymes. Amino acids Asp120, Cys-221, and His-263, presumably involved in the binding of the second zinc ion in subclass B2 MBLs, were also conserved in the PFM-like proteins.

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FIG 2

Dendrogram of PFM-1, PFM-2, and PFM-3 in comparison with representative class B β-lactamases subjected to neighbor-joining analysis. The alignment used for the tree calculation was performed with the Clustal Omega program. Numbers in parentheses indicate percentages of amino acid identity with PFM-1. The β-lactamases used for the comparisons (GenBank accession numbers) were Sfh-1 (NZ_AUZV01000091.1), CphA-1 (X57102), ImiS (Y10415), ImiH (AJ548797), VIM-1 (AJ278514), IMP-1 (EF027105), NDM-1 (KJ018857), POM-1 (EU315252), and PAM-1 (AB858498). Percentages of amino acid identities compared to PFM-1 are indicated.

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FIG 3

Alignment of the amino acid sequences of subclass B2 MBLs. Residues conserved in the enzymes are indicated by asterisks; colons indicate conservation between groups with strongly similar properties; dots indicate conservation between groups with weakly similar properties. The BBL numbering scheme (in bold) is used for residues conserved in MBLs.

In order to gain insight into the β-lactam resistance phenotype conferred by the corresponding proteins, the bla_PFM-1, bla_PFM-2, and bla_PFM-3 genes of P. synxantha strain MCP-106, P. libanensis strain CIP105460, and P. fluorescens PF1 were cloned into plasmid pTOPO (Invitrogen, Illkirch, France) and expressed in Escherichia coli. Cloning experiments were performed using the pCR-blunt TOPO cloning kit (Invitrogen, Illkirch, France) after amplification of the genes with primers PFM-1-Fw and PFM-1-Rv for bla_PFM-1 and with primers PFM-2-Fw and PFM-2-Rw for bla_PFM-2 and bla_PFM-3. The resulting recombinant plasmids were transformed into chemically competent E. coli TOP10 strains. Once expressed in E. coli TOP10, similar resistance phenotypes were observed with the different PFM variants, with reduced susceptibility to carbapenems seen (Table 1) but paradoxically no effect on the other β-lactams tested such as amoxicillin, ticarcillin, cefoxitin, cefotaxime, and ceftazidime (data not shown). MICs of carbapenems were determined by Etest and showed values for the PFM-3-producing recombinant strain that were higher than those obtained with the PFM-1-producing and PFM-2-producing recombinant strains, particularly for imipenem (Table 1).

Purification of the PFM-1 enzyme was performed using a four-liter LB broth culture of E. coli TOP10 (pTOPO-bla_PFM-1) recombinant strain supplemented with kanamycin (50 μg/ml) and inoculated for 24 h at 37°C under shaking conditions. The bacterial culture was centrifuged, and the pellet was resuspended in Tris-HCl buffer (50 mM Tris-HCl, 100 μM ZnCl₂, pH 8.5) and sonicated using a Vibra-Cell 75186 sonicator (Thermo Fisher Scientific). After filtration using a 0.22-μm-pore-size nitrocellulose filter, the crude extract was loaded in a Q-Sepharose column connected to an ÄKTAprime chromatography system (GE Healthcare, Glattbrugg, Switzerland) and eluted with a linear NaCl gradient. The presence of the β-lactamase was monitored using the Rapid Carba NP test (19), and the fractions showing the highest β-lactamase activity were pooled and dialyzed against 100 mM phosphate buffer (pH 7.0), prior to 10-fold concentration performed with a Vivaspin 20 concentrator (GE Healthcare). The purified β-lactamase extract was immediately used for enzymatic determinations.

The protein concentrations were measured using Bradford reagent (Sigma-Aldrich, Buchs, Switzerland), and the purity of the enzyme was estimated by SDS-PAGE analysis (GenScript, NJ, USA). The purity of PFM-1 was estimated to be >95%, with a single dominant band visible on the SDS-polyacrylamide gel. Kinetic measurements were performed at room temperature using phosphate-buffered saline (PBS) buffer (0.1 M, pH 7) supplemented with ZnSO₄ (5 μM) using a UV/visible Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences, Buckinghamshire, United Kingdom). This kinetic analysis confirmed that PFM-1 hydrolyzed carbapenems; however, the catalytic efficiency was slightly lower than that seen with the previously described subclass B2 MBLs (Table 2). In contrast, hydrolysis of other β-lactam substrates such as benzylpenicillin or cefotaxime was not detected (k_cat value < 0.01 s⁻¹). This study therefore characterized a novel family of subclass B2 MBLs with substantial carbapenemase activity. Compared to other subclass B2 MBLs, PFM-1 hydrolysis is limited to carbapenems, and the catalytic efficiency is lower.

The levels of G+C content of bla_PFM-1 (50%) and bla_PFM-2/-3 (52%) differed from the expected range of the G+C content of Pseudomonas genes (ca. 60%); in addition, the fact that no other bla_PFM-like genes were identified in several fully sequenced genomes of P. fluorescens strains available in the GenBank databases further suggests a non-Pseudomonas origin. However, no obvious genetic element that could have been involved in the acquisition of that gene was observed in its nearby genetic environment. Similarly, no mobile genetic elements were identified in their upstream vicinity by analyzing the genes showing significant identities with bla_PFM-1 in the GenBank database. It may be speculated that those genes have been acquired by transformation since P. fluorescens strains, as with many other Gram-negative nonfermenters, are spontaneously transformable at high frequency (28). However, a discrepancy was always noticed between all of the putative MBL-encoding genes (including bla_PFM-1) and the surrounding chromosomal sequences in term of GC content (ca. 50% versus ca 60%), suggesting a foreign origin (data not shown).

This work underlines that P. fluorescens-like species may possess class B β-lactamase genes that are, however, not systematically present in their genomes. Although strains belonging to the P. fluorescens complex are rarely involved in human infections, they are widely disseminated in the environment and parts of the human microbiota and can also be found in chicken meat (16). Those bacterial species may therefore constitute reservoirs of antimicrobial resistance genes (29).