Aliidiomarina shirensis as Possible Source of the Integron- and Plasmid-Mediated Fosfomycin Resistance Gene fosC2

ABSTRACT

In-silico analysis and cloning experiments identified a fosC2-like fosfomycin resistance gene in the chromosome of Aliidiomarina shirensis, with our data suggesting that this bacterium might be added to the list of species identified as reservoirs of fos-like genes that were subsequently acquired by other Gram-negative species. Indeed, the fosC2 gene was identified as acquired in Providencia huaxinensis and Aeromonas hydrophila isolates, with this gene being located in class 1 integron structures in the latter cases. Biochemical characterization and site-directed mutagenesis showed a higher catalytic efficiency for the intrinsic FosC2^AS (from A. shirensis) than for the acquired FosC2 (from P. huaxinensis) enzyme due to a single substitution in the amino acid sequence (Gly43Glu). Notably, this study constitutes the first identification of the likely natural reservoir of a complete gene cassette (including its attC site).

INTRODUCTION

Considering the increasing rates of resistance to fluoroquinolones and broad-spectrum cephalosporins that is observed worldwide in Enterobacterales, the broad-spectrum bactericidal antibiotic fosfomycin is now a drug of choice for treating uncomplicated urinary tract infections (UTIs), being often used as first-line oral therapy (1, 2). The use of fosfomycin also contributes to reducing the selection pressure resulting from the frequent use of carbapenems to treat urinary tract infections caused by isolates producing extended-spectrum β-lactamases (ESBL) (3, 4). Therefore, the preservation of fosfomycin efficacy is gaining importance and requires surveillance studies aimed to evaluate the spread of, and identify the origin and dissemination of, fosfomycin resistance determinants.

Acquisition of fosfomycin resistance may be related to different mechanisms, including reduction of drug uptake due to mutations in the genes encoding GlpT and UhpT transporters and mutations in the fosfomycin target: the enzyme catalyzing the first step in peptidoglycan biosynthesis, namely, MurA. Among these, transporter mutations are the most frequent fosfomycin resistance mechanisms (5). Some Fos metalloenzymes are transferable resistance mechanisms, responsible for inactivating fosfomycin by catalyzing the opening of the epoxide ring from fosfomycin molecules (5). Although there is a family of kinase enzymes (namely, FomA and FomB) produced by fosfomycin-producing microorganisms (Streptomyces and Pseudomonas syringae), these are not considered clinically relevant (6). In pathogenic Gram-negative bacteria, fosfomycin inactivation is mediated by metalloenzymes that catalyze the opening of the epoxide ring by adding glutathione (FosA1-FosA10, FosL1-L2, FosC2), bacillithiol (FosB), or H₂O (FosX). Glutathione-S-transferases are the most prevalent fosfomycin resistance determinants in Enterobacterales (5). The dissemination of the corresponding genes mainly involves plasmids, transposons, and integrons.

Several natural reservoirs of the fos resistance genes have been already identified, corresponding to several Gram-negative species, including Enterobacter cloacae for fosA1 and fosA2; Kluvvera georgiana for fosA3 and fosA4; Klebsiella pneumoniae for fosA5, fosA6, and fosA10; Leclercia adecarboxylata for fosA8; and Klebsiella variicola for fosA9 (7). In contrast, the natural reservoirs of the more recently reported fos resistance genes (fosL1, fosL2, and fosC2) remain unknown (8, 9).

The fosC2 gene was first reported as plasmid-located (of an unknown incompatibility group) in Escherichia coli, and subsequently reported on an IncL/M plasmid type in a single Enterobacter cloacae isolate from China (9, 10), being associated with a class 1 integron structure in the latter case. Notably, FosC2 shares only 72% amino-acid identity with FosC previously identified in Achromobacter xylosoxydans, with 66% identity between the fosC2 and fosC genes at the nucleotide level. By performing an in silico analysis, we also identified the fosC2 gene in the genome of two other strains, namely, a single Providencia huaxinensis and a single Aeromonas hydrophila strain. In the latter two cases, the gene was also located as part of a class 1 integron.

Our objective was to identify the progenitor of the fosC2 gene and to understand its mobilization pathway.

RESULTS

Origin of the fosC2 gene.

In-silico analysis identified a gene putatively encoding a FosC2-like protein in the chromosome of the Idiomarinaceae species Aliidiomarina shirensis, in a strain isolated from Shira Lake in Khakasia (GenBank accession no. NZ.PIPP00000000.1). This deduced protein sequence shared a high percentage of amino-acid identity (96%, four-amino-acids difference) with FosC2, and was thereafter named FosC2^AS. Since the latter sequence was identified in the unique genome sequence of that species, as available in the database, it was not possible to speculate further about the intrinsic nature of this gene in that species. Nevertheless, since no obvious mobile genetic element was identified in the vicinity of the fosC2^AS gene, it might be speculated that this gene is intrinsic to that species.

Of note, another genome of A. shirensis is currently available in the database (GenBank JAGYK001), corresponding to strain M30B69, in which no fosC2-like gene was identified. However, this genome sequence is only partial and no formal conclusion can be drawn.

Interestingly, in silico analysis also revealed that the fosC2 gene was identified in the genomes of two sequenced strains, being identified within class 1 integron structures as a form of gene cassette. These fosC2-bearing class 1 integrons were identified in the chromosome of a Providencia huaxinensis strain (GenBank accession no. CP031123.2) and on a plasmid of an Aeromonas hydrophila strain (pSS332-218k; accession no. CP071152.1), both recovered from rectal swabs of two patients from China (11–13). It is noteworthy that the fosC2 gene had already been reported as part of class 1 integron structures, being part of multidrug-resistant plasmids (9).

Phylogenetic analysis performed with FosC2 and the other FosA-like amino acid sequences revealed that FosC2 was distantly related to the FosA enzymes, representing a distinct cluster (Fig. 1). FosC2 shares 56% of amino acid sequences with FosA3, but possessed conserved residues of FosA3 proteins that are implicated in dimer formation of the FosA3 proteins, in Mn²⁺ and K⁺ binding, and in fosfomycin binding (14) (Fig. 2). Although significant differences exist between FosA3 and FosC2 in terms of protein sequences, both enzymes were shown to modify fosfomycin through glutathione transferase activity (9).

FIG 1.

Phylogenetic tree obtained for the FosA1-8, FosL1-2, and FosC2 proteins identified by distance method using the neighbor-joining algorithm (SeaView version 4 software). Branch lengths are drawn to scale and are proportional to the number of amino acid substitutions with 500 bootstrap replications. Distance along the vertical axis has no significance. Species known as the natural reservoirs of these fosfomycin-modifying enzymes are indicated.

FIG 2.

Amino-acid alignment of plasmid-encoded Fos proteins. Red, blue, green, and purple boxes bracket amino acids of the dimer interface loop and those implicated in Mn²⁺ coordination, in the K⁺ binding loop, and in fosfomycin binding, respectively (14).

Genetic context of the fosC2 gene.

To fully elucidate the genetic context of the fosC2 gene, comparative genomic analysis was performed using the fosC2-flanking regions from the five fosC2-associated sequences identified in the database. Analysis of the whole-genome sequence of A. shirensis showed that the fosC2^AS gene was surrounded by genes encoding hypothetical proteins, including AraC (transcriptional regulator) and SDR (oxidoreductase). As mentioned previously, no obvious mobile genetic element was found in the fosC2^AS-flanking regions. Nevertheless, analysis of P. huaxinensis genome sequences showed that the fosC2 gene was located within a chromosomal class 1 integron surrounded by insertion sequences (IS26) which also carried several resistance genes, such as aac-(6′)-Ib, ere(A), bla_OXA-21, dfrA1, and sulA. In E. cloacae, the fosC2 gene was also located in a class 1 integron structure, along with the carbapenemase gene bla_IMP-34 gene, with the integron located on an IncL/M-plasmid type; the integron also carried a complete set of tni module (tniR, tniQ, tniB, and tniA), with the tniA gene being truncated by IS26 (10). By analyzing the genome sequence of the A. hydrophila strain, we again identified the fosC2 gene in a plasmid-borne class 1 integron, along with other resistance genes, including aac(6′)-Ib, bla_OXA-1, and catB (13). In E. coli, the first fosC2 gene described in 2010 by Wachino et al. was the first gene cassette located in a class 1 integron accompanied by dfrA17 and aadA5, with the integron gene truncated by an insertion sequence (IS26) (Fig. 3A).

FIG 3.

(A) Genetic structures surrounding the fosC2^AS gene on the chromosome of A. shirensis, the fosC2 gene on the chromosomal integron of P. huaxinensis, the fosC2 gene on the plasmid of E. cloacae, the fosC2 gene on the plasmid of A. hydrophila, and the fosC2 gene on the plasmid of E. coli. The fragment of the sequence shared by A. shirensis, P. huaxinensis, E. cloacae, and A. hydrophila is bounded by dashed lines. Sequences in the beginning and at the end of the shared section belonging to the attC structure are shown. Red letters indicate mismatches between different species. (B) attC architecture. Double-stranded circular form of the fosC2AS cassette showing the sequence of the attC site. The direction of the fosC2^AS gene is indicated by a horizontal arrowhead and the start and stop codons on the top strand are in bold and underlined. Core sites are underlined and labeled (1L, 2L, 1R, 2R), with red letters indicating the mismatches in the 1L and 2L core site. The position at which the cassette opens up to give the linear form is shown by a vertical arrow.

Accordingly, the sequences located closely upstream and downstream from the fosC2 genes in P. huaxinensis and E. cloacae shared significant identity at the nucleotide level (98%) with those in A. shirensis (Fig. 3A). By analyzing the fosC2^AS gene surrounding sequences, the core site 1R (TTATGTT) of the attC recombination site was identified upstream from fosC2^AS. Downstream from the fosC2^AS gene, the rest of the attC site was identified, containing the 1L, 2L, and 2R core sites (Fig. 3A). According to previous works on that topic, we may therefore speculate that a circularization of the surrounding sequences encompassing the fosC2^AS gene can be elaborated under a double-stranded circular form, leading to a fosC2^AS gene cassette which includes the resistance gene and its attC recombination site, which is 75 bp in size (Fig. 3B).

By analyzing the attC recombination site in detail, two simple sites composed of a pair of “core sites” were identified, corresponding to 1L and 2L, and 2R and 1R, respectively. The fosC2^AS gene cassette contained a central region, corresponding to the distance between 1L/2L and 2R/1R pairs, which was 37 bp in length (15, 16) (Fig. 3B).

The GTT triplet, which is part of the 1R core site and known to be a key factor in the activity of the attC sites, was fully conserved when comparing fosC2^AS-associated sequences from A. shirensis, P. huaxinensis, E. cloacae, and A. hydrophila. As previously reported, the recombination site is located between the G and the first T of 1R (17). After the recombination process, a linear integrated form of the cassette may open up at this position; the 1R is subsequently found at the start of the cassette, separated from the rest of the attC site by the gene cassette (Fig. 3A).

Functional characterization of FosC2 and FosC2^AS enzymes.

In order to characterize the fosfomycin resistance conferred by FosC2 and FosC2^AS enzymes, the two corresponding fos genes together with other Fos-encoding genes (belonging to different groups), including fosA3, fosA8, and fosL1, were cloned into the pUCp24 recipient vector, followed by expression in an Escherichia coli TOP10 background. A disk diffusion test showed that the FosC2- and FosC2^AS-producing E. coli recombinant strains, respectively, exhibited different inhibition diameters around the fosfomycin disk, with the FosC2^AS-producing E. coli recombinant strain showing the highest level of resistance. Determination of MIC values showed high levels of resistance (>256 μg/mL) for all the E. coli recombinant strains, with the exception of the FosC2-producing E. coli recombinant strain (128 μg/mL) (Table 1).

TABLE 1.

MICs of fosfomycin for the E. coli recombinant strains producing different Fos enzymes

E. coli strain	Fosfomycin MIC (μg/mL)
TOP10	<4
TOP10 (fosA3)	>256
TOP10 (fosA8)	>256
TOP10 (fosL1)	>256
TOP10 (fosC2)	128
TOP10 (fosC2AS)	>256

These data suggest that several of the amino acid substitutions present in the FosC2 protein sequence likely play a critical role in the increased activity of the enzyme. A comparative analysis of the FosC2^AS and FosC2 amino acid sequences was performed in silico using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo/) (Fig. S1). FosC2 and FosC2^AS differed by four amino acid substitutions (Ile5Leu, Leu19Val, Ser33Arg, and Gly43Glu). Among those substitutions, Glu43 was found to be unique to FosC2 when compared to all other Fos sequences (Fig. 2). Therefore, we hypothesized that Glu43 was involved in the reduced glutathione transferase activity of FosC2 compared to that of the other Fos proteins analyzed here.

To further confirm this hypothesis, we performed site-directed mutagenesis. Hence, the Glu43 amino acid of FosC2 enzyme was substituted for a Gly residue that is present in the FosC2^AS enzyme. The fosfomycin resistance phenotype of the FosC2^AS-producing E. coli recombinant strain was recovered in the FosC2^Glu43Gly-producing E. coli recombinant strain, confirming our previous hypothesis (Fig. S2).

DISCUSSION

We report here the identification of an integron- and plasmid-mediated fosfomycin resistance determinant, FosC2, in P. huaxinensis and A. hydrophila. The FosC2 enzyme was shown to confer resistance to fosfomycin, but to a lesser extent compared with the other acquired Fos-type enzymes identified so far, namely, FosA1-10 and FosL1-2. Site-directed mutagenesis experiments showed that Gly43 was a critical residue for the catalytic efficiency of FosC2, and is likely critical for Fos enzymes in general. This amino acid residue may indeed be related to a glutathione-binding site described, based on the position of fosfomycin in the active site, and encompassing the residues Tyr39, Trp46, and Cys48, as suggested (18).

The FosC2-producing E. coli recombinant strain showed an MIC value for fosfomycin which was similar to the clinical intravenous breakpoint (32 μg/mL), despite the corresponding gene being expressed at a high level in this strain due to the cloning context (strong promoter, high-copy-number plasmid); this further highlights that the fosC2 gene might confer only reduced susceptibility among clinical isolates, thus possibly remaining undetected, consequently favoring the silent spread of this gene. In silico analysis showed that this gene had actually been acquired by a different bacterial species which had previously been recovered exclusively in China.

A. shirensis was identified here as the possible natural reservoir of the fosC2 gene, and could therefore be added to the list of Gram-negative bacteria that naturally possess a fos-like gene, from which such a resistance gene might “escape” and eventually be acquired by other Gram-negative species. However, formal confirmation of the intrinsic presence of such a gene in other A. shirensis genomes will be required by investigating additional strains of that species (which are unfortunately not available at the present stage), since it is not possible to exclude the hypothesis that the fosC2-like gene cassette could have been acquired by A. shirensis.

Of note, the GC% content of both the fosC2^AS gene and the Aliidiomarina shirensis AIS whole-genome sequence were very similar (46.45% and 45%, respectively), reinforcing the hypothesis that fosC2^AS could be intrinsic to this species.

This finding further highlights the indirect interplay between environmental and clinical bacteria in terms of antibiotic resistance, a feature that is currently seriously considered under the “One-Health” concept. Notably, the fosC2 gene, when acquired, was always part of a class 1 integron structure, being located at either a chromosomal or plasmid position class 1 integron genetic element. To the best of our knowledge, this might be the very first identification of a gene cassette origin. This discovery therefore contributes to a better understanding of the process leading to gene cassette mobilization, specifically with respect to the origin and mobilization of fosfomycin resistance genes that might often be related to integron features, as highlighted by previous studies (19, 20).

In fact, different models of cassette genesis have been suggested. In the 1990s, with a very limited amount of genetic data available, Recchia and Hall hypothesized that antibiotic resistance gene cassettes are likely to be assembled from separate pools of genes and attC sequences (16, 21–23). Subsequently, IIC-attC intron-encoded reverse transcriptases were described as having both RNA-dependent and DNA-dependent polymerase activities. These data suggested a model of gene cassette formation in which a transcriptional terminator of the RNA of the gene was fused to an attC site by IIC-attC intron using homologous recombination and then retrotranscribed into a DNA gene cassette (24, 25). Here, we provide evidence for an original structure showing that the resistance gene and its attC site structure could be found associated in the chromosome of a given bacterial species, likely corresponding to their progenitor. In A. shirensis, both the fosC2 resistance gene and its associated attC site were found perfectly assembled in the chromosome, shedding new light on the gene cassette genesis process.

Considering that such context might constitute a very good model, future work will be performed in vitro to further investigate the process of gene cassette acquisition by class 1 integrons.

MATERIALS AND METHODS

Bacterial strains and plasmids.

E. coli TOP10 reference strains was used as recipients for cloning experiments. P. huaxinensis GDMCC1.1382, belonging to the Morganellaceae spp. family, was isolated from a human rectal swab at the Guangdong Microbiology Culture Centre, China (12). A. shirensis JCM17761, a fosfomycin-resistant Gram-negative bacterium recovered from Shira Lake (Republic of Khakassia, Russia), was obtained from the Japan Collection of Microorganisms, Japan (11). Plasmid pUCp24 (26) was used as the shuttle vector for cloning and expression in E. coli TOP10 recipient strains. The list of strains and plasmids used in the study is shown in Table 2.

TABLE 2.

Strains and plasmids used in this study

Strain or plasmid	Relevant genotype or phenotype	Reference or source
Strains
E. coli TOP10	Recipient strain for cloning experiments	Invitrogen
P. huaxinensis GDMCC 1.1382	Fosfomycin-resistant Morganellaceae (human rectal swab); integron-encoded fosC2 gene	(12)
A. shirensis JCM17761	Fosfomycin resistant Gram-negative species (Shira Lake); chromosomal-encoded fosC2AS gene	(11)
Plasmids
pUCp24	Shuttle vector; gentamycin resistant	(26)
pFosA3	Recombinant plasmid pUCp24 with fosA3	This study
pFosA8	Recombinant plasmid pUCp24 with fosA8	This study
pFosL1	Recombinant plasmid pUCp24 with fosL1	This study
pFosC2	Recombinant plasmid pUCp24 with fosC2	This study
pFosC2AS	Recombinant plasmid pUCp24 with fosC2AS	This study

Fosfomycin resistance determination.

To assess fosfomycin susceptibility, the agar dilution method using Mueller-Hinton agar (MHA, reference 64884; BioRad, Marnes-la-Coquette, France) supplemented with 25 μg/mL glucose-6-phosphate was used as recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines (27). The breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used as a reference (28).

FosA-like role in fosfomycin resistance.

The contribution of a potential FosA-like enzyme in fosfomycin resistance was assessed by using the disk diffusion method as described by Nakamura et al. (29). Briefly, the fosfomycin-resistant isolates used in this work were inoculated onto Mueller-Hinton agar plates supplemented with 25 μg/mL of glucose-6-phosphate, at an ∼0.5 McFarland standard. Two disks containing 200 μg of fosfomycin, with and without 0.5 mg of the FosA inhibitor sodium phosphonoformate (PPF) (Sigma-Aldrich), were added to the plates, which were incubated at 35 ± 2°C overnight. PPF selectively inhibits FosA-like proteins, allowing for differentiation between plasmid-mediated FosA and other mechanisms of fosfomycin resistance. An increase of ≥5 mm in the diameter of the growth inhibition zone in the presence of PPF was interpreted as FosA-related resistance. E. coli strain 249 producing the plasmid-encoded FosL1 enzyme was used as the positive control (8).

Molecular analyses.

DNA was extracted from P. huaxinensis and A. shirensis using the QIAamp DNA Mini Kit and the QIAcube workstation (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. PCR amplification and sequencing (Microsynth, Balgach, Switzerland) was performed to detect and confirm the presence and sequence of integron-mediated fosC2 genes using the primers named fosC2, listed in Table S1 in the supplemental material. PCR conditions were 95°C for 5 min followed by 30 cycles at 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s, with a final extension of 72°C for 5 min. Sequences were analyzed using CloneManager Professional (Sci-Ed Software, Denver, CO, USA) (30).

Whole-genome DNA of the fosC2-positive isolates was downloaded from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). The resulting genomes were analyzed in order to describe the different genetic contexts for the fosC2 gene found in the database. Genetic environment surrounding the fosC2 genes and their schematic representations was performed using SnapGene Viewer 4.0 (GSL Biotech LLC, IL, USA).

The fos genes from E. coli (fosA3, fosA8, and fosL1), A. shirensis (fosC2^AS), and P. huaxinensis (fosC2) were cloned into the pUCp24 shuttle-vector by using primers listed in Table S1 and transformed in E. coli TOP10. Hence, all of these fos genes were expressed under the control of the same promoter provided by plasmid pUC24.

site-directed mutagenesis.

The substitution of the Glu43 of FosC2 by the Gly43 residue was performed using a Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA), using primers [Fw (5′-CTG AGC TTG GGG GCT ACT TGG ATT TG-3′)/Rv (5′-ATA TGC CCC ACT ATC CCA-3′)] generated by the NEBaseChanger online tool (http://nebasechanger.neb.com/) using pUCP24-fosC2 recombinant plasmid as the template, following the manufacturer’s recommendations. The mutant constructs were ligated and transformed into the E. coli TOP10 background.

Data availability.

The WGS data of the isolates analyzed in this work were obtained from the GenBank database under the following accession numbers: Aliidiomarina shirensis AIS^T (NZ.PIPP00000000.1), Providencia huaxinensis WCHPr000369^T (CP031123.2), pIMP-HB623 plasmid from Enterobacter cloacae CRE623 (KM877517.1), and pSS332-218k plasmid from Aeromonas hydrophila (CP071152.1).