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Plasmid-Borne AFM Alleles in Pseudomonas aeruginosa Clinical Isolates from China

ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a pathogen of global concern due to the fact that therapeutic drugs are limited. Metallo-β-lactamase (MBL)-producing P. aeruginosa has become a critical part of CRPA. Alcaligenes faecalis metallo-β-lactamase (AFM) is a newly identified subclass B1b MBL. In this study, 487 P. aeruginosa strains isolated from patients and the environment in an intensive care unit were screened for AFM alleles. Five AFM-producing strains were identified, including four AFM-2-producing strains (ST262) and one AFM-4-producing strain (ST671). AFM-2-producing strains were isolated from rectal and throat swabs, and AFM-4-producing strains were isolated from the water sink. The blaAFM-2 carrying plasmids belonged to the IncP-2 type, while the blaAFM-4 carrying plasmid pAR19438 was a pSTY-like megaplasmid. Plasmid pAR19438 was acquired blaAFM-4 by the integration of the Tn1403-like transposon. All blaAFM genes were embedded in an ISCR29-blaAFM unit core module flanked by class 1 integrons. The core module of blaAFM-2 was ISCR29-ΔgroL-blaAFM-2-bleMBL-ΔtrpF-ΔISCR, while the core module of blaAFM-4 was ISCR29-ΔgroL-blaAFM-2-bleMBL-ΔtrpF-ISCR-msrB-msrA-yfcG-corA-ΔISCR. The flanking sequences of ISCR29-blaAFM units also differed. The expression of AFM-2 and AFM-4 in DH5α and PAO1 illustrated the same effect for the evaluation of the MICs of β-lactams, except for aztreonam. Identification of AFM-4 underscores that the quick spread and emerging development of mutants of MBLs require continuous surveillance in P. aeruginosa. IMPORTANCE Acquiring metallo-β-lactamase genes is one of the important carbapenem resistance mechanisms of P. aeruginosa. Alcaligenes faecalis metallo-β-lactamase is a newly identified metallo-β-lactamase, the prevalence and genetic context of which need to be explored. In this study, we identified AFM-producing P. aeruginosa strains among clinical isolates and found a new mutant of AFM, AFM-4. The blaAFM-4 carrying plasmid pAR19438 was a pSTY-like megaplasmid, unlike the plasmids encoding other blaAFM alleles. The genetic context of blaAFM-4 was also different. However, AFM-2 and AFM-4 had the same impacts on antibiotic susceptibility. The presence and transmission of AFM alleles in P. aeruginosa pose a challenge to clinical practice.

In this study, we screened AFM alleles from 487 P. aeruginosa strains isolated from an intensive care unit (ICU) in Zhejiang Provincial People's Hospital in Zhejiang, China. Five AFM-producing P. aeruginosa strains were identified, including one strain carrying a novel AFM allele AFM-4. The genetic characteristics of these strains were analyzed and the effect of AFM-4 on antibiotic resistance was evaluated.

RESULTS
Identification of AFM-carrying strains. We collected 487 P. aeruginosa isolates from environmental and patient samples in the ICU of Zhejiang Provincial People's Hospital, between April and July 2021. We screened these isolates for AFM alleles and consequently found five AFM-carrying P. aeruginosa strains, named AR19438, AR19640, AR19726, AR19727, and AR23664. AR19438 was isolated from the surface of a water sink in the ward. The other specimens were isolated from the same patient within 45 days: AR19640 and AR19726 were isolated from rectal swabs, and AR19727 and AR23664 were isolated from throat swabs. Genome sequencing revealed that AR19438 belonged to sequence type (ST) 671 and that the other four strains belonged to ST262. Using PCR and sequencing, we identified AR19438 as a novel AFM allele, named AFM-4 (GenBank accession: OM049002), while the other strains had AFM-2. In the amino acid sequence of AFM-2, the valine 15 residue is replaced with alanine (GenBank accession: AYV97588), while in the amino acid sequence of AFM-4, the proline 13 residue is replaced with leucine (Fig. S1).
Results of antimicrobial susceptibility testing. For antimicrobial susceptibility testing, we included another three relative strains without AFM for comparison (Table 1). AR19437 belonging to ST671 was isolated from the scupper of the water sink, and on the same day, AR19438 was isolated. AR19538 and AR23663 were isolated from rectal swabs of the same patient from whom the other ST262 strains were isolated.
Both AR19437 and AR19438 are susceptible to aztreonam, amikacin, gentamicin, and colistin. AR19438 is resistant to meropenem, imipenem, cefepime, piperacillin, ceftazidime, piperacillin-tazobactam, ceftazidime-avibactam, and ciprofloxacin, while AR19437 showed various susceptibility to these antibiotics. All AFM-2-carrying strains were resistant to most tested antibiotics except for aztreonam and colistin, and the latter was active against all  Table 2). The characteristics of AFM-carrying plasmids. Whole-genome sequencing revealed that bla AFM-2 and bla AFM-4 were embedded in two distinct plasmids. As indicated by alignment, all of the AFM-2 carrying strains contained plasmids that were quite similar to the sole plasmid pAR19640 in AR19640 (Fig. 1). Plasmid pAR19640 shares 90% and 80% query coverage with pNDTH9845 (GenBank accession: CP073081) and pWTJH17 (GenBank accession: CP073083), respectively. The backbones of these homologous plasmids were similar to those of the IncP-2 plasmid pOZ176, indicating that they belonged to the same megaplasmid family (9). The plasmid pAR19640 is 49562 bp long with an average GC content of 56%. There are numerous resistance genes embedded in pAR19640, which can roughly be divided into two gene clusters. One resistance gene cluster located from approximately 25 kbp to 55 kbp contains most of the resistance genes, including bla AFM-2 . The other resistance gene cluster contains only eight predicted open reading frames flanked by IS1394 and IS6100 (approximately 480 kbp). The differences among pAR19640, pNDTH9845 and pWTJH17 are linked to the presence of insertion sequences (ISs) or genes encoding integrase, indicating that these plasmids evolved through multiple insertions and recombinations.
AFM-4 carrying plasmid pAR19438 is 275,369 bp long with an average GC content of 56%. Among several homologous plasmids found in NCBI, pAR19438 is most similar to pSTY (GenBank accession: CP003962), which is a megaplasmid conserved by Pseudomonas taiwanensis strain VLB120 that renders solvent tolerance (10,11). pAR19438 shares 75% query coverage and 99.98% identity with pSTY by BLAST. The common parts of the two plasmids encode the type IV secretion system (T4SS) and plasmid maintenance proteins. However, there were three long-segment insertions/deletions between pAR19438 and pSTY (Fig. 2). The first deletion in pAR19438 begins with xerD encoding phage integrase site specific recombinase and ends approximately 41 kbp downstream. This part of the sequence includes genes encoding enzymes such as 2-keto-4-pentenoate hydratase (mphD) and toluene efflux pump gene cluster ttgGHI. The second deletion in pAR19438 includes mercury-tolerant operon (mer) and phenylacetic acid degradative pathway genes (paa genes) and there are two Tn3-family transposase genes (tnpA) close to both ends. These two gene segments in pSTY provide VLB120 with solvent tolerance. The last difference is the long-segment insertion in pAR19438 compared to pSTY, which consists of abundant mobile genetic elements and antimicrobial resistance genes, including bla AFM-4 . This long-segment insertion includes two transposons, Tn1403-like transposon and Tn4662a, with a 163 bp gap between them (Fig. 2). Tn1403-like transposon is 31,682 bp in length flanked by 7 bp direct repeats (59-ctctctg-39) including bla AFM-4 and its surrounding sequences, which are concretely shown in Fig. 3. Tn4662a is 5,635 bp in length flanked by 7 bp direct repeats (59-aacatgg-39) including genes encoding DNA invertase, RelE/ParE family toxin, helix-turn-helix protein, etc.
We used oriTfinder to identify four components of a conjugative apparatus in pAR19438 and pAR19640, and only identified the T4SS in pAR19438 (Fig. 2). We conducted conjunction Plasmid-Borne AFM Microbiology Spectrum assays to determine the transferability of pAR19640 and pAR19438. The experiments were performed three times but all failed, suggesting that these plasmids are not conjugative. Genetic contexts of bla AFM alleles in P. aeruginosa. All AFM alleles have been reported in P. aeruginosa strains thus far (7,8). We chose pNDTH9845, pWTJH17, pHS17-127 (GenBank accession: CP061377), and two AFM-carrying plasmids in this study to compare the genetic contexts of AFM alleles in P. aeruginosa (Fig. 3).
The genetic contexts of bla AFM-2 and bla AFM-3 are both an ISCR29-bla AFM unit flanked by two complex class 1 integrons indicates. The ISCR29-bla AFM units of bla AFM-2 and bla AFM-3 sequentially consist of ISCR29, DgroL, bla AFM , ble MBL , DtrpF, and DISCR. In pAR19640, the downstream region of the ISCR29-bla AFM unit is part of an In4-like class 1 integron, ISCR1 and ISEC28. The In4-like class 1 integron contains resistance genes, including sul1, aadA, cmlA, aadB, and aac(6")-II. In pNDTH9845, aadA was disrupted by IS1394. In pWTJH17, aadB was missing and the region downstream of ISCR1 was further replaced by ISPpu30, sul1, tniB, and gnat. The upstream part of the ISCR29-bla AFM unit in pAR19640 is a Tn1403-derived class 1 integron, which is identical to pNDTH9845. This integron contains resistance genes, including sul1, dfrA27, arr-3, bla OXA-246 , cmlA8, and aacA4. The upstream part in pWTJH17 is different from that of pAR19640 in that an ISPre2 has been inserted in the intergenic region between arr-3 and bla OXA-246 , and tnpA-tnpR has been replaced by IS26-induced insertion carrying msr(E)-mph(E) (macrolide resistance genes). The genetic context of bla AFM-4 is significantly different from those of bla AFM-2 and bla AFM-3 . First, the core module ISCR29-bla AFM-4 unit is identical to the unit in pHS17-127, in  which there is a 4998 bp insertion of putative ISCR with the cassette array msrB-msrA-yfcG-corA. Second, the 7091 bp-long sequence downstream of the ISCR29-bla AFM unit is coincidentally identical to part of pAR19640 (ranging from 478504 bp to 485435 bp), except for the 163 bp-long deletion at 61 bp upstream of IS6100. This region includes the cassette array sulP-uspA-dksA-yiiK, which can be found in Tn1404 (GenBank accession: AH008062) and Tn6609 (GenBank accession: MZ361367). Finally, the upstream region of the ISCR29-bla AFM unit is a different Tn1403-derived class 1 integron from pAR19640, since they share the same transposase and 39 conserved sequences but contain different passenger segments. The passenger segments include dfrA42 (trimethoprim resistance gene), abc (gene encoding probable ATP-binding/permease fusion ABC transporter), fepC (gene encoding ferric enterobactin transport protein), qnrVC6 (quinolone resistance gene), and two insertion sequences (ISCR1 and IS26). In bla AFM-1 carrying pHS17-127, the resistance gene cassette array upstream of the ISCR29-bla AFM-1 unit consists of aacA4, bla IMP-45 , bla OXA-1 , catB3, sul1, qnrVC6, and bla PER-1 .
Effects of AFM-4 on the MICs of b-lactams. To better understand the novel gene bla AFM-4 conferring b-lactam resistance, we cloned bla AFM-4 into the plasmid pGK1900 and then transformed it into E. coli DH5a and P. aeruginosa PAO1. The effects of AFM-4 differed in DH5a and PAO1 (Table 3).
In DH5a, AFM-4 increased the MIC levels up to 2-4-fold for meropenem, imipenem, cefepime, piperacillin and piperacillin-tazobactam. The MICs of ceftazidime and ceftazidimeavibactam were elevated over 8-fold by AFM-4. In PAO1, the MICs of all b-lactams increased over 5-fold, except aztreonam, which of course cannot be hydrolyzed by AFM-4, an MBL. We cloned bla AFM-2 with the same method. The antimicrobial susceptibility testing results showed that there was little difference in the antibiotic resistance effects between AFM-2 and AFM-4.

DISCUSSION
As a novel MBL, AFM alleles have not been widely disseminated in P. aeruginosa. In multicenter surveillance, only three AFM-producing P. aeruginosa strains were identified from 605 clinical isolates, accounting for 0.50% (8). In our study, AFM-producing strains occupied 1.03% (5/487) of all isolated strains, and four strains isolated from the same patient (out of five AFMproducing strains) had clonal relatedness. AFM-2-producing P. aeruginosa strains belong to ST262 and AFM-4-producing P. aeruginosa AR19438 belongs to ST671, both of which are not common clinical sequence types of CRPA. Compared to the strains without AFM, AFM-producing strains showed higher MICs of b-lactams, fluoroquinolones, and aminoglycosides, as a result of acquiring drug resistant plasmids. To date, ST671 P. aeruginosa has been reported to contaminate rinse water, leading to the bronchoscope-related pseudo-outbreak (12). AR19438 was also isolated from the sink surface, indicating that ST671 P. aeruginosa isolates might be related to the water environment of wards. The contamination of the water environment was closely associated with P. aeruginosa infections in ICUs (13)(14)(15). Thus, we should be alerted to the dissemination of the AFM-4-producing ST671 P. aeruginosa clone in the ICU.
In this study, bla AFM-2 genes were observed to be encoded on plasmids similar to those previously reported in other P. aeruginosa strains (7,9). However, bla AFM-4 was located on a pSTY-like megaplasmid, which was different from the IncP-2 plasmids encoding bla AFM1-3 in P. aeruginosa strains (7,8). Comparing pAR19438 to pSTY, pAR19438 may have acquired bla AFM-4 by the integration of the AFM-4-encoding Tn1403-like transposon. Abbreviations are the same as in Table 2.

Plasmid-Borne AFM Microbiology Spectrum
Only seven pSTY-like megaplasmids were found in the NCBI database, all belonging to Pseudomonas sp. (Table S1), indicating the species specificity of this megaplasmid family. Except for pSTY, other pSTY-like megaplasmids have all been identified in China in recent years. Continuous surveillance of these newly emerged megaplasmids is required due to their ability to be integrated with resistance genes and spread among Pseudomonas sp. ISCRs are linked with variant resistance determinates and are transmitted by rolling circle, which contributes to the rapid dissemination of drug resistance (16,17). The genetic context of bla AFM is an ISCR29-bla AFM unit core module flanked by class 1 integrons. ISCR29-bla AFM units are identical in most bla AFM1-3 carrying plasmids, suggesting that ISCR29 is responsible for the dissemination of bla AFM . However, in pAR19438, this core module was interrupted by ISCR-msrB-msrA-yfcG-corA, which added to its complexity. Although the AFM-producing P. aeruginosa strains were isolated in a single room, the genetic contexts of bla AFM-2 and bla AFM-4 varied dramatically, indicating that there was no horizontal transfer of drug resistance genes between strains of the two sequence types.
The simulated structure of AFM was a classic ab/ba sandwich similar to all MBLs, with Zn1 interacting with histidine residues 117, 119 and 186 and Zn2 interacting with residues Asp121, Cys205 and His247 (7). The mutation sites of AFM-2, AFM-3 and AFM-4 compared to AFM-1 are Pro13 or Ala15, and these sites have no impact on either the overall fold or the active sites (Fig. S2), which could explain the close impact of AFM-2 and AFM-4 on antimicrobial susceptibility.
In conclusion, we identified AFM-producing P. aeruginosa strains in the ICU and analyzed their genetic characteristics. A novel AFM allele, AFM-4, was identified, which was located in the pSTY-like plasmid and embedded in a different genetic context compared to other AFM alleles in P. aeruginosa. The continuous emergence of variant carbapenemase-producing Pseudomonas aeruginosa needs to be properly addressed. And persistent genomic surveillance should be conducted, as carbapenemase genes are widespread.

MATERIALS AND METHODS
Isolation and identification of AFM-carrying strains. P. aeruginosa strains were isolated from environmental and patient samples in an ICU in Zhejiang Provincial People's Hospital in Zhejiang Province, China using Pseudomonas Isolation Agar plates (18). Then we used a Vitek II automatic microbe analysis instrument (bioMérieux) to identify the species. AFM alleles were screened using PCR (PCR) to amplify the full ORF with the primer pairs: AFM-F (59-cagctctgagattgaggcga-39) and AFM-R (59-attgggtgtgacgtggtca-39). PCR products were sequenced by Sanger sequencing.
bla AFM cloned into P. aeruginosa and E. coli. bla AFM genes and their upstream sequences (a total of 1711 bp) were amplified from AR19438 and AR19640 and cloned into pGK1900 using a Hieff Clone Plus One Step Cloning kit (7). The AFM expressing plasmids were transformed into E. coli DH5a by chemical transformation and into P. aeruginosa PAO1 by electroporation transformation.
Conjugation of plasmids. Conjugation experiments were performed with AR19438 and AR19640 as donors and a rifampicin-resistant mutant of PAO1 as the recipient (18). The antibiotics and the concentration used for selection were rifampicin (300 mg/mL) and meropenem (8 mg/mL).
Data statement. The data in our research are available to access. The genome sequences of P. aeruginosa strains in this study have been deposited in NCBI under the BioProject (Accession: PRJNA826241). The AFM-4 allele has been deposited in NCBI with the accession number NG_079223.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1.5 MB.