ABSTRACT
The Bacteriophage Exclusion (BREX) system is a novel antiphage defense system identified in Bacillus cereus in 2015. The purpose of this study was to investigate the presence of the BREX system defenses against antibiotic-resistant plasmids such as blaKPC and blaNDM invasion in Escherichia coli. The BREX system was present in 5.4% (23/424) of E. coli clinical isolates and 6.5% (84/1283) of E. coli strains with completely sequenced genomes in the GenBank database. All 23 BREX-positive E. coli clinical isolates were susceptible to carbapenems, while all five isolates carrying blaKPC and 11 carrying blaNDM were BREX-negative. For E. coli strains in the GenBank database, 37 of 38 strains carrying blaKPC and 109 of 111 strains carrying blaNDM were BREX negative. The recognition site sequence of methyltransferase PglX in a clinical E. coli 3756 was 5′-CANCATC-3′ using PacBio single-molecular real-time sequencing. The transformation efficiency of plasmid psgRNA-ColAori-target with the PglX recognition site was reduced by 100% compared with the plasmid without the recognition site in E. coli DH5α-pHSG398-BREX. The BREX showed lower defense efficacy against plasmid psgRNA-15Aori-target which had the same plasmid backbone but different surrounding sequences of recognition sites with psgRNA-ColAori-target. The conjugation frequency of the KPC-2 plasmid and NDM-5 plasmid in E. coli 3756-ΔBREX was higher than that in E. coli 3756 clinical isolate (1.0 × 10−6 vs 1.3 × 10−7 and 5.5 × 10−7 vs 1.7 × 10−8, respectively). This study demonstrated that the type I BREX system defends against antibiotic-resistant plasmids in E. coli.
KEYWORDS: BREX system, carbapenem resistance, plasmids, Escherichia coli
INTRODUCTION
Escherichia coli is the main pathogen causing bacteremia, urinary tract infection, biliary tract infection, and other clinical infections (1). Carbapenem-resistant Enterobacterales, which are associated with high mortality rates, are a worldwide health threat and are listed as high-priority pathogens by the World Health Organization (2). blaKPC and blaNDM are the main carbapenemase genes in Klebsiella pneumoniae and E. coli, respectively. These carbapenemase genes are usually carried by large conjugative plasmids that can horizontally transfer between different species and strains (3). Bacteria have evolved multiple defense systems under long-term selection pressure to protect themselves from infection by viruses and other invading DNA through horizontal gene transfer (HGT) (4). Conjugation by plasmids, transduction by bacteriophages, and natural transformation by extracellular DNA are the three primary mechanisms of HGT in bacteria, contributing significantly to the rapid spread of resistance (5).
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are adaptive immune systems consisting of cas genes and CRISPR loci (6). These systems can defend against invaders by targeting and cleaving the protospacers in the respective invader genomes. Previous studies showed that the type I-E CRISPR-Cas system could effectively influence the invasion and stability of blaKPC-IncF plasmids in K. pneumoniae. The scarcity of CRISPR-Cas systems is one of the potential factors leading to the propagation of blaKPC-IncF plasmids in clonal group 258 (CG258) (7). However, the CRISPR-Cas system in E. coli appears to differ from that in K. pneumoniae. Our previous study showed that the CRISPR-Cas system in E. coli is unable to target blaKPC-producing plasmids due to the absence of matching protospacers, while the type I R-M system in E. coli could impede blaKPC-carrying plasmid conjugation (8).
PglZ, a putative member of the alkaline phosphatase superfamily enriched within defense islands, is essential for the phage growth limitation (Pgl) system in Streptomyces coelicolor (9). Further research in 2015 revealed that pglZ-domain genes are embedded in a gene cluster encoding a novel defense system named the Bacteriophage Exclusion (BREX) system, which is present in about 10% of bacterial and archaeal genomes (10). This system can balance the need to acquire beneficial transfer events through HGT with the need to defend against infection by bacteriophages. Based on gene sequence, content, and number, the BREX system is classified into six subtypes. The type I BREX system is the predominant type in E. coli. The type I BREX system is encoded by a six-gene cassette which encodes a SAM-dependent methyltransferase (pglX), a putative alkaline phosphatase (pglZ), a putative ATPase (brxC), a putative Lon-like protease (brxL), a putative DNA-binding protein (brxA), and a protein of unknown function (brxB) (10, 11). BREX is a novel defense mechanism that involves DNA methylation of the bacterial genome to distinguish self from non-self. The BREX system of E. coli methylates the fifth residue of the non-palindromic 5′-GGTAAG-3′ motif in the bacterial genome (12). However, limited previous research on the BREX system focused on defense against bacteriophages. Whether the existence of a BREX system could block the invasion of antimicrobial resistance plasmids in bacteria remained unclear.
The aims of the present study were (i) to investigate the prevalence of the BREX system in clinical isolates of E. coli and publicly available complete E. coli genome sequences and (ii) to determine whether the BREX system could hinder the invasion of antimicrobial resistance plasmids in E. coli.
RESULTS
Distribution and the genetic environment of the BREX system in E. coli
BREX systems were detected in 5.4% (23/424) of E. coli clinical isolates collected at Huashan Hospital in 2017 and in 6.5% (84/1283) of E. coli strains with complete genome sequences in the GenBank database (Table S1). To analyze gene sequence conservation, we performed an alignment of BREX system gene clusters. Among the 23 BREX-positive clinical isolates, 22 had brxA, brxB, brxC, pglZ, and brxL genes and had a high sequence similarity with E. coli HS (NC_009800.1) carrying the type I BREX system, showing 97%–100% identity, while 3.6 kb gene pglX had a low homology. The identities of the initial 1.9 kb and the final 800 bp were 86%–92% and 87%–98%, respectively, and the middle parts of the gene sequences could not be matched (Table S2). The pglX of 72/84 BREX-positive E. coli strains in GenBank and one clinical isolate of E. coli 3756 with whole-genome sequencing could be clustered using CD-HIT-EST, resulting in 17 clusters. Analysis of the genomic environment surrounding the BREX system revealed that 58.9% (43/73) of the BREX system were located on the downstream of nanCMS operon involved in sialic acid catabolism to provide a source of carbon and nitrogen in E. coli (13), while the downstream of the BREX system diversified containing variety hypothetical proteins (Fig. S1; Table S3). The BREX-positive clinical isolate E. coli 1080 matched the type I BREX system on plasmid pEREF in E. fergusonii ATCC 35469.
In all, 10 sequence types were identified among the 23 clinical E. coli isolates carrying a BREX system, with ST131 (56.5%, 13/23) being the dominant one, followed by ST1531 (8.7%, 2/23). The most prevalent sequence type among the 84 strains in GenBank was ST16 (11.9%, 10/84), followed by ST131 and ST155 (8.5%, 8/84).
Correlation between the absence of a BREX system and the presence of blaKPC and blaNDM
The prevalence of blaKPC in E. coli clinical isolates and the GenBank database was 1.2% (5/424) and 3.0% (38/1283), respectively. For the blaNDM genes, the detection rate in E. coli clinical isolates and the GenBank database was 2.6% (11/424) and 8.7% (111/1283), respectively. All E. coli clinical isolates carrying blaKPC (n = 5) and blaNDM (n = 11) were BREX negative. For E. coli strains in the GenBank database, 37 of 38 carrying blaKPC and 109 of 111 carrying blaNDM were BREX negative. All 23 clinical isolates and 81 of 84 strains in the GenBank database with BREX-positive were negative for blaKPC and blaNDM.
The antimicrobial resistance rates to piperacillin, ertapenem, cefoperazone-sulbactam, and piperacillin-tazobactam were significantly lower in 23 BREX-positive E. coli clinical isolates than in 401 BREX-negative isolates (Table 1). These observations suggest that the BREX system may influence blaKPC and blaNDM gene dissemination in E. coli.
TABLE 1.
Comparison of antimicrobial resistance between BREX-positive and BREX-negative E. coli clinical isolates
| BREX positive ( = 23), n (%) | BREX negative ( = 401), n (%) | P | |
|---|---|---|---|
| Amikacin | 0 (0) | 28 (7) | 0.386 |
| Aztreonam | 8 (35) | 218 (54) | 0.085 |
| Cefotaxime | 16 (70) | 290 (72) | 0.811 |
| Cefoxitin | 3 (13) | 132 (33) | 0.063 |
| Ceftazidime | 10 (43) | 239 (60) | 0.133 |
| Cefoperazone-sulbactam | 0 (0) | 113 (28) | 0.001 |
| Cefepime | 6 (26) | 188 (47) | 0.055 |
| Ciprofloxacin | 17 (74) | 333 (83) | 0.261 |
| Ertapenem | 0 (0) | 63 (16) | 0.034 |
| Imipenem | 0 (0) | 40 (10) | 0.150 |
| Meropenem | 0 (0) | 43 (11) | 0.150 |
| Piperacillin | 15 (65) | 332 (83) | 0.047 |
| Piperacillin-tazobactam | 0 (0) | 82 (20) | 0.011 |
| Polymyxin | 0 (0) | 0 (0) | >0.999 |
| Tigecycline | 0 (0) | 21 (5) | 0.618 |
| Trimethoprim-sulfamethoxazole | 10 (43) | 215 (54) | 0.393 |
Statistically significant correlations (P < 0.05) are shown in bold font.
Among the 1,283 E. coli with complete genomes, 84 BREX-positive E. coli strains had fewer plasmids compared with 1,199 BREX-negative strains (median n = 2, IQR 1–13 vs median n = 3, IQR 1–24, P = 0.593); however, there was no statistically difference. BREX-positive E. coli contained fewer mob markers which are associated with distinct plasmid mobility types, compared with E. coli without the BREX system (median n = 2, IQR 0–14 vs median n = 2, IQR 0–20, P < 0.001) among the 1,283 complete and 27,963 draft E. coli genome sequences. A similar result was also observed that the BREX-positive strains contained less rep markers (median n = 4, IQR 0–17 vs median n = 4, IQR 0–18, P < 0.001) and predicted plasmids DNA (median n = 104,738, IQR 1,020–627,265 vs median n = 110,998, IQR 1,001–5,635,318, P < 0.001) than BREX-negative strains (Table S4). The data above revealed that BREX-positive strains have fewer numbers and predicted DNA of plasmids.
Identification of target sequences of the BREX system and its presence in KPC and NDM plasmids
The SMRT sequencing of E. coli 3756 and E. coli 3756-ΔBREX were performed to identify the methylation sites of the BREX system. Sequencing of E. coli 3756 DNA showed that 99.99% of the 6589 genomic 5′-CANCATC-3′ sites were modified, with methylation observed at the adenine base in the fifth position of the motif, while no modification was observed in the E. coli 3756-ΔBREX (Fig. 1). Further plasmid transformation assays were performed in E. coli DH5α-pHSG398-BREX to examine this recognition site using two plasmids psgRNA-ColAori without the recognition site and psgRNA-ColAori-target containing the recognition site. The transformation efficiency of plasmid psgRNA-ColAori-target was reduced by 100% compared with plasmid psgRNA-ColAori.
Fig 1.
The methylation site of BREX in genomic DNA of clinical E. coli 3756. The arrow shows the site of BREX-dependent methylation.
Further analysis was performed to identify the distribution of the recognition site among all plasmids (n = 8,068) in E. coli from the NCBI database. 92.4% (7,455/8,068) of plasmids contained the target sequence of the BREX system. The prevalent plasmid types among the 7,455 BREX-targeted plasmids were IncFIB (26.3%, 1,958/7,455), followed by IncFII (14.0%, 1,046/7,455). The distribution of common β-lactams resistance genes in BREX-targeted plasmids were as follows: blaKPC (1.0%, 77/7,455), blaNDM (3.8%, 283/7,455), blaOXA (4.4%, 328/7,455) with blaOXA-48 (10.7%, 35/328), blaCTX-M (10.4%, 777/7,455), and blaTEM (15.6%, 1,165/7,455), etc. All blaKPC-producing plasmids and blaNDM-positive plasmids contained this recognition site, suggesting that they could be potentially blocked by the BREX system.
BREX impeded the transfer of blaKPC and blaNDM plasmids
A conjugation assay was performed to determine whether the BREX system could impede the transfer of KPC and NDM plasmids in E. coli. The mean conjugation frequency of clinical E. coli 3756-ΔBREX (1.0 × 10−6) was higher than that of E. coli 3756 (1.3 × 10−7) (Fig. 2A) and a similar result was observed in E. coli DH5α-pHSG398 and E. coli DH5α-pHSG398-BREX (3.8 × 10−1vs. 3.9 × 10−2) (Fig. 2B) when using E. coli J53 with blaKPC-2-IncC plasmid (250 CANCATC motifs) as a donor strain.
Fig 2.
Comparison of conjugation frequency of blaKPC-2 and blaNDM-5 plasmids. (A) Comparison of conjugation frequency of blaKPC-2 from the donor strain E. coli J53 to the recipient strain in the presence (E. coli 3756) or absence (E. coli 3756-ΔBREX) of a BREX system. (B) Conjugation frequency of blaKPC-2 from the donor strain E. coli J53 to the recipient strain in the presence (E. coli DH5α-pHSG398-BREX) or absence (E. coli DH5α-pHSG398) of a BREX system. (C) Comparison of conjugation frequency of blaNDM-5 from the donor strain E. coli J53 to the recipient strain E. coli 3756 or E. coli 3756-ΔBREX. (D) Conjugation frequency of blaNDM-5 from the donor strain E. coli DH5α to the recipient strain E. coli DH5α-pHSG398-BREX or E. coli DH5α-pHSG398. ****P < 0.0001 between the two groups as determined using the chi-squared test.
The mean conjugation frequency of the blaNDM-5-IncX3 plasmid in clinical E. coli 3756-ΔBREX (5.5 × 10−7) was higher than that of E. coli 3756 (1.7 × 10−8) (Fig. 2C) when using the E. coli J53-blaNDM-5 with 46 CANCATC motifs as a donor strain (conjugator from clinical isolate of E. coli 4), and a similar result was also observed in E. coli DH5α-pHSG398 and E. coli DH5α-pHSG398-BREX (1.9 × 10−1 vs 1.9 × 10−2) (Fig. 2D) using the E. coli DH5α-blaNDM-5 (the plasmid transformed from clinical isolate of E. coli 6, this plasmid contains with 134 CANCATC motifs) as a donor strain.
The surrounding genetic environment of BREX recognition motifs may dictate the response of BREX system
The transformation efficiencies of plasmids psgRNA-ColAori-target and psgRNA-15Aori-target were reduced by 100% and 17% compared with plasmid psgRNA-ColAori and psgRNA-15Aori, respectively (Fig. 3). The psgRNA-ColAori-target and psgRNA-15Aori-target plasmids encoded the same number of BREX recognition motif (two motifs) and shared the same plasmid backbone but had different surrounding sequences of the two recognition sites (Fig. 4). The plasmid psgRNA-ColAori-target had lower transformation efficiency comparing with psgRNA-15Aori-target indicating that the surrounding environment of the recognition motif may influence the defense effects of BREX system.
Fig 3.
(A) Comparison of transformation efficiency of psgRNA-ColAori and psgRNA-ColAori-target plasmids in DH5α-pHSG398-BREX. (B) Comparison of transformation efficiency of psgRNA-15Aori and psgRNA-15Aori-target plasmids in DH5α-pHSG398-BREX.
Fig 4.
Alignment of surrounding sequences of two recognition sites in psgRNA-ColAori-target and psgRNA-15Aori-target plasmids. The two recognition sites of the BREX system are shown in the arrow.
DISCUSSION
This study demonstrates for the first time that the type I BREX system impedes the spread of blaKPC and blaNDM plasmids in E. coli. We found that all E. coli clinical isolates with a BREX system were susceptible to carbapenems, while all isolates carrying blaKPC or blaNDM were BREX negative. 37 of 38 KPC-positive and 109 of 111 NDM-positive E. coli strains in the GenBank database were BREX negative. All blaKPC and blaNDM plasmids in E. coli from the GenBank database carried the recognition site of the BREX system, indicating the potential function of the BREX system in preventing the acquisition of blaKPC and blaNDM plasmids. The conjugation assay showed that the BREX system was able to impede the transmission of blaKPC and blaNDM plasmids.
In the conjugation experiment using E. coli, DH5α-blaNDM-5 (plasmid transformed from clinical isolate E. coli 6) as a donor strain and E. coli 3756 and 3756-ΔBREX as the recipients were unsuccessful after three repeated conjugation assays. This might be because the E. coli 3756-ΔBREX and E. coli 3756 strains were not suitable recipients for this NDM-5 plasmid. For example, morphologic features of the strain may affect cell wall permeability and consequently impair conjugation (14). The conjugation was successful when we used the E. coli J53-blaNDM-5 (conjugator from a clinical isolate of E. coli 4) as a donor strain.
The exchange of foreign DNA, such as plasmids, viruses, and transposons, occurs among different species of bacteria and is driven by HGT (15). Bacteria have evolved a multitude of defense systems to prevent invasion by bacteriophages and other mobile genetic elements such as antimicrobial resistance plasmids. Defense systems such as the type I-E CRISPR-Cas system and the type I R-M system are probable factors affecting the entry of blaKPC plasmids in K. pneumoniae and E. coli, respectively (8, 16). BREX, a phage resistance system first described in 2015, is genetically and phenotypically different from previously characterized phage defense mechanisms (17).
Based on associated gene combinations, BREX systems are classified into six subtypes, composed of four to eight associated genes appearing in a highly conserved order (Fig. S2). The type I BREX system, the most abundant type, is present in about 55% of BREX-containing genomes and is comprised of six core genes (brxA, brxB, brxC, pglX, pglZ, and brxL) (18). The similarity of type I BREX system gene clusters between BREX-positive clinical E. coli isolates and E. coli HS showed that brxA, brxB, brxC, pglZ, and brxL are highly homologous, while the similarity of pglX is not very high. The recognition sites of BREX systems in clinical E. coli 3756, E. fergusonii ATCC35469, E. coli HS, and Salmonella enterica sv Typhimurium were CANCATC, GCTAAT, GGTAAG, and GATCAG, respectively (19), suggesting that the biological role of DNA methylation may be strain specific, which was consistent with low similarity of pglX. There are also important commonalities—they are asymmetric, and adenine in the fifth position of the recognition site was methylated.
The conjugation frequencies were much lower than those of transformation efficiencies in this study. One of the reasons is the presence of anti-BREX protein in clinical plasmids. It was reported that the Ocr protein of the T7 phage, a DNA mimic protein, could abrogate the protective function of the BREX system by binding to DNA methyltransferase PglX (20). We searched for Ocr and its 10 homologs protein in blaKPC-2-IncC and blaNDM-5-IncX3 plasmids used in conjugation experiments. No candidates of Ocr and its 10 homologs anti-BREX protein were detected (data not shown), whereas it does not rule out the possibility of additional genes on plasmids offsetting the anti-plasmid effects of BREX.
Recent studies have found that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage while the scale of BREX response does not correlate to a number of BREX motifs. Phages CS16 and Mav have only six SNPs between them but vary in BREX response, suggesting SNPs may dictate BREX defense phenotypic change. Plasmids psgRNA-ColAori-target and psgRNA-15Aori-target shared the same backbone of plasmid and encoded the same number of BREX recognition motifs (two motifs). The plasmid psgRNA-ColAori-target had lower transformation efficiency compared with psgRNA-15Aori-target indicating that the surrounding environment of the recognition motif may influence the degree of defense effects of the BREX system which was consistent with a previous study (19).
BREX is a largely uncharacterized defense system that methylates a specific asymmetric DNA site in bacterial DNA to distinguish self from non-self, which is similar to the R-M system. However, a few lines of evidence including blocked phage DNA replication, non-degradation of phage DNA, and non-palindromic methylation sites suggest that BREX is not a simple R-M system (10). A study reported that a multidrug-resistant plasmid contains interplaying BREX and BrxU (type IV restriction enzyme) defensive systems. BrxU can target DNA containing modified cytosines (including 5-methyl cytosine, 5-hydroxymethyl cytosine, and glucosyl-5-hydroxymethyl cytosine modifications) that cannot be recognized by the BREX system (21). This “belt and braces” approach ensures better protection for the bacterial host. In the present study, the BREX system and BrxU gene on the plasmid of clinical E. coli isolate 1080 was highly similar to the type I BREX system and BrxU gene cluster on plasmid pEFER in E. fergusonii ATCC 35469.
The distribution of BREX systems in other Gram-negative bacteria, the ability to hinder the transmission of other antimicrobial resistance plasmids, and the defense mechanism against resistance plasmids need to be studied further to improve our understanding of BREX systems and their potential clinical value.
MATERIALS AND METHODS
Bacterial strains
A total of 424 unduplicated clinical isolates of E. coli were collected from Huashan Hospital in 2017. E. coli J53-blaKPC-2 and E. coli J53-blaNDM-5 were obtained by conjugating blaKPC-2 and blaNDM-5 plasmids into E. coli J53, respectively, from clinical isolates of E. coli 1496 and E. coli 4. E. coli DH5α-blaNDM-5 was obtained by transforming the blaNDM-5 plasmid into E. coli DH5α via an electroporation method from a clinical isolate of E. coli 6. These strains were used as donors in the conjugation experiments. The genome sequences of isolates above have been deposited in GenBank under BioProject PRJNA1059248. All publicly available E. coli complete genome sequences (29246 in total, including 1,283 complete genome sequences) and complete sequences of 8,068 plasmids were downloaded from the NCBI genome database in May 2023.
Prevalence of BREX systems, blaKPC and blaNDM
PCR analysis was performed to screen for the pglZ, blaKPC, and blaNDM in E. coli clinical isolates. Primers are listed in Table S5. Subsequently, BREX-harboring E. coli isolates were subjected to next-generation sequencing. BLAST+ software (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST) was used to detect the BREX system, blaKPC and blaNDM in 1,283 E. coli strains with complete genome sequencing. The pglX of BREX-positive strains could be clustered using CD-HIT-EST.
Analysis of multilocus sequence typing (MLST), plasmid typing, and resistance genes
MLST was performed on 23 BREX-positive clinical E. coli isolates with next-generation sequencing and 1,283 publicly available E. coli strains using MLST software (https://github.com/tseemann/mlst). The plasmid type and resistance genes of plasmids in E. coli were identified by PlasmidFinder2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) and ResFinder2.1 (https://cge.cbs.dtu.dk/services/ResFinder/). MOB-suite was used to identify plasmids rep and mob markers (22).
Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents were determined with the agar dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) (23). MICs of tigecycline and polymyxin were determined with broth microdilution. Polymyxin MICs were interpreted using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) criteria and for tigecycline, the Food and Drug Administration (FDA) breakpoints were used (24, 25).
BREX system cluster deletion in E. coli
Homologous recombination was used to knock out the BREX cluster in clinical E. coli isolate 3756. Briefly, the plasmid pMD18T-hyg-BREX-upstream-downstream with the upstream and downstream homologous arms of BREX were constructed using primers listed in Table S5. Then λRed recombination and Flp-FRT were used to construct a BREX-deletion variant (3756-∆BREX). Colonies with BREX deletion were verified by PCR using the six-gene primers listed in Table S5.
Pacific biosciences sequencing
The methylome of clinical E. coli 3756 and its BREX-deletion variant 3756-∆BREX were sequenced using PacBio SMRT sequencing technology to identify the methylation status. Sequencing was performed on either a PacBio sequel II. SMRTLink_12.0.0 software and Base Modification Analysis for Sequel II data were performed to identify DNA modifications and their corresponding target motifs.
Plasmid transformation assays
The pMB1 replicon of psgRNA (without the recognition sequence 5′-CANCATC-3′) was removed by inverse PCR. The ColA and p15A replicon were amplified using 2 × Phanta Flash Master Mix High-Fidelity DNA polymerase with primers containing a 15–22 base overlap listed in Table S5 and then cloned on psgRNA using the in-fusion cloning method. The psgRNA-ColAori-target and psgRNA-15Aori-target were constructed through cloning amplified ColA replicon and p15A replicon sequences containing PglX recognition sequence 5′-CAT(G)CATC-3′ into psgRNA, respectively.
Chemically competent DH5α-pHSG398-BREX cells were prepared with the Ultra-Competent cell prep kit (Sangon, No. B529303) and used as recipients. Aliquots of cells (100 µL) were transformed with 1,000 ng of psgRNA-ColAori, psgRNA-ColAori-target, psgRNA-15Aori, or psgRNA-15Aori-target plasmid DNA, diluted in 900 µL Luria broth, and incubated for 1 h at 37°C for recovery. Transformants were selected on LB agar plates containing both chloramphenicol (50 µg/mL) and spectinomycin hydrochloride (75 µg/mL). Transformation assays were performed at least three times. Transformation efficiency was defined as the number of transformants per nanogram of the plasmid.
Conjugation assay
Clinical isolates E. coli 3756 and E. coli 3756-ΔBREX were used as recipients with E. coli J53-blaKPC-2 and E. coli J53-blaNDM-5 as the donor strains (conjugator from clinical isolate of E. coli 1496 and 4, respectively). The donors and recipients were cultured to the logarithmic phase, mixed in a 1:1 ratio, and spotted on LB agar plates, followed by incubation at 37°C for 20 h. Transconjugants were selected on LB agar plates containing both ciprofloxacin (8 µg/mL) and meropenem (0.5 µg/mL) or ciprofloxacin (4 µg/mL) and piperacillin-tazobactam (64/4 µg/mL). LB agar plates supplemented with only ciprofloxacin (8 µg/mL or 4 µg/mL) were used to calculate the total number of recipients. Conjugation frequencies were calculated by dividing the number of transconjugants by the total number of recipients and were determined in three independent tests.
Due to the unsuccessful complementation of E. coli 3756-ΔBREX with a BREX system, E. coli DH5α-pHSG398-BREX was constructed. A 14 kb fragment containing the entire set of six BREX genes from the clinical isolate E. coli 3756 was amplified by PCR (primers are listed in Table S5) and then ligated into pHSG398. pHSG398 and pHSG398-BREX were transformed into E. coli DH5α lacking an endogenous BREX system. The successful construction strains were selected with plates containing chloramphenicol (50 µg/mL) and verified by PCR sequencing.
E. coli DH5α-pHSG398 and E. coli DH5α-pHSG398-BREX were used as recipients with E. coli J53-blaKPC-2 as a donor strain. Due to the donor strain, E. coli J53-blaNDM-5 conjugated from a clinical isolate of E. coli 4 was resistant to chloramphenicol (Table S6); therefore, transconjugant could not be selected with chloramphenicol and ampicillin. E. coli DH5α-blaNDM-5 obtained by electroporation from a clinical isolate of E. coli 6 was used as a donor. Transconjugants were selected on LB agar plates containing both chloramphenicol (50 µg/mL) and ampicillin (100 µg/mL). LB agar plates with chloramphenicol (50 µg/mL) were used to calculate the total number of recipients.
Statistical analysis
Statistical significance was determined using the chi-squared test, Fisher exact test, and Wilcoxon rank-sum test. Differences were considered statistically significant at P < 0.05.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (grant numbers 81991531 and 81991530) and the Shanghai Municipal Science and Technology Commission (grant number 22410710300).
Contributor Information
Yuan Yuan, Email: lanzhouyy@163.com.
Minggui Wang, Email: mgwang@fudan.edu.cn.
Alessandra Carattoli, Universita degli studi di roma La Sapienza, Viale del Policlinico, Roma, Italy.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/aac.01128-23.
17 clusters of BREX positive strains.
Classification of BREX subtypes.
The type I BREX system positive Escherichia coli strains.
Blast results of BREX system gene cluster.
Sequence clustering of BREX-positive Escherichia coli strains.
Measures of plasmid number, diversity and load among E. coli with complete and draft genomes.
Primers used in this study.
Antimicrobial susceptibility profiles of Escherichia coli strains used in this study.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
17 clusters of BREX positive strains.
Classification of BREX subtypes.
The type I BREX system positive Escherichia coli strains.
Blast results of BREX system gene cluster.
Sequence clustering of BREX-positive Escherichia coli strains.
Measures of plasmid number, diversity and load among E. coli with complete and draft genomes.
Primers used in this study.
Antimicrobial susceptibility profiles of Escherichia coli strains used in this study.