Unearthing New ccr Genes and Staphylococcal Cassette Chromosome Elements in Staphylococci Through Genome Mining

Abstract

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen capable of causing a spectrum of infections, from superficial skin abscesses to severe cases such as pneumonia, osteomyelitis, and septicemia. A defining characteristic of MRSA is its antibiotic resistance, with the methicillin-resistant determinant mecA consistently located within a mobile genetic element SCC (staphylococcal cassette chromosome), SCCmec [1, 2]. These elements are highly diverse, with lengths typically ranging from 21 to 67 kb, and are often present in tandem to form composite islands that can exceed 100 kb [1, 2]. In addition to carrying mecA, SCC/SCCmec elements can encode a variety of virulence factors and resistance determinants, thereby enhancing the tolerance of S. aureus to environmental stresses, such as antimicrobials, heavy metals, polyamines, and acids [3–5.] Consequently, the typing of SCC/SCCmec has been a focus in molecular epidemiological studies, aiming to understand the global emergence and spread of MRSA. An International Working Group on the Staphylococcal Cassette Chromosome elements (IWG-SCC) was established to provide consensus guidelines on this nomenclature (https://www.sccmec.org/index.php/en/).

Ccr recombinase plays a crucial role in the excision and integration of the direct repeat sequences at the terminals of SCC/SCCmec elements, generating extrachromosomal circular intermediates (ciSCC) that facilitate interbacterial transmission [1, 2]. SCCmec sequence typing is primarily based on various combinations of ccr gene complexes and mec gene complexes. To date, 5 mec gene complexes have been identified as types A, B, C1, C2, and E [1, 2]. According to the guidelines of the IWG-SCC, ccr genes are categorized into ccrA, ccrB, and ccrC [1]. Typically, the genes encoding CcrA and CcrB are located in tandem and function jointly, whereas CcrC functions independently [6]. As of now, 8 ccrA, 9 ccrB, and 2 ccrC have been reported in the past 2 decades [7–10.] These types of ccr form 10 ccr gene complexes, in combination with 5 mec gene complexes, form at least 15 different types of SCCmec elements (I–XV). In addition to S. aureus, SCCmec elements as well as ccr genes can also be found in other staphylococci as well as mammaliicocci, such as Staphylococcus epidermidis [5], Staphylococcus haemolyticus [4], and Mammaliicoccus sciuri (formerly Staphylococcus sciuri) [11].

Therefore, the type of ccr genes plays a crucial role in the typing of SCC/SCCmec elements. In this study, we followed the guidelines of IWG-SCC and discovered 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) by searching the nucleotide total species database of the National Center for Biotechnology Information (NCBI). These ccr genes were combined to form 12 novel ccr gene complexes. Two novel combinations between the novel ccr and the mec complexes were indicated, including ccrA9B3 and class A mec gene complex, as well as ccrA10B1 and class B mec gene complex. The 2 novel combinations have not yet been found in S. aureus, and so they will not be identified as novel SCCmec types according to the IWG-SCC guidelines until similar combinations are found in S. aureus.

METHODS

Screening Novel ccr Genes based on Nucleotide Database

To screen for novel ccr genes in a nucleotide database, a representative gene for each reported type of ccrA, ccrB, and ccrC genes were selected [3, 12]. This resulted in 3 representative gene libraries consisting of 8 sequences of ccrA1–ccrA8, 9 sequences of ccrB1–ccrB9, and 2 sequences of ccrC1–ccrC2. The Basic Local Alignment Search Tool with default parameters (ncbi-blast-2.13.0+-win64.exe, the parameters used are all default) were used to query the NCBI nucleotide (nt) total species database, which was downloaded using IBM Aspera Connect software (Supplementary Material File 1). This initial round of screening aimed to identify sequences that were similar to the representative genes in the libraries. In the first round of screening, the parameters were set as “-outfmt6 -evalue 1e-5 -num_alignments 1 -max_targetq-seqs 10″ to retrieve the most similar sequences. Next, the VLOOKUP function was used to search for sequences with nucleotide similarity ranging from 50% to 85% to all representative genes in the libraries. This second round of screening aimed to identify the most closely related sequences while excluding false-positive matches.

The second round of screening produced a set of candidate sequences that were aligned with each other using DNAMAN software to remove duplicate sequences (the parameters were “Full Alignment and Try both strands”). The Conserved Domains database in NCBI with default parameters was used to confirm that the candidate ccr genes belong to a serine recombinase family. To reject false-positive results, the candidate ccr genes were aligned with 5 randomly selected allotypes of their most similar ccr genes separately. The sequence identities less than 85% were considered as acceptable matches. Finally, the novel ccr genes that passed all the screening steps were designated according to the rules of the IWG-SCC committee [1].

Identification of Novel ccr Gene Complexes and Novel SCCmec/SCC Elements

A comprehensive analysis was conducted to identify novel ccr gene complexes and SCCmec/SCC elements. First, the whole-genome sequences of strains containing the novel ccr genes were obtained from the NCBI nucleotide database. The ccr gene clusters were then analyzed using the IWG-SCC method to determine the ccr gene complexes [1]. To determine the length of novel SCCs, a direct repeat universal sequence (NGANGCNTANCANAANTNA) was used as a query sequence to identify the conserved direct repeat sequences from both upstream and downstream of the novel ccr gene complexes. If the novel SCC elements contain the mecA gene, they were designated according to IWG-SCC rules. Finally, the SCCmec/SCC element was visually represented using the Easyfig_2.2.5 software to provide a clear and concise schematic representation. Overall, this approach allowed us to successfully identify and characterize novel ccr gene complexes and SCCmec/SCC elements.

Bioinformatics Analysis

To further elucidate the phylogenetic relationships between the identified ccr gene complexes, we referred to published ccr gene phylogenetic trees [1, 9], 1–3 allotypes of all ccr genes for analysis. DNAMAN and iTOL (https://itol.embl.de/) were used to display the phylogenetic relationships, with an analysis method of Observed Divergency and a parameter Bootstrap of 2000. To investigate the distribution of the novel ccr genes, we included the nucleotide sequences of ccrA9 (SL13), ccrA10 (UTI-042y), ccrA11 (2794_1), ccrA12 (LTH3730), ccrA13 (GDH8C110P), ccrA14 (NCTC13838), ccrB10 (HKUOPL8), ccrB11 (NCTC13838), ccrB12 (2794_1), ccrC3 (FDAARGOS_538), ccrC4 (Tienloo1), and ccrC5 (WC28) as query sequences for BLAST comparison with the NCBI nr/nt database (default parameters). This approach allowed us to gain insights into the distribution and diversity of the identified ccr gene complexes and their potential role in promoting antimicrobial resistance.

Function Determination of the Novel Ccr Recombinase

To investigate the ability of novel Ccr recombinases to excise the SCCmec element, polymerase chain reaction (PCR) assays were performed according to the method described previously [13]. Briefly, in our previous study, a SCCmec-deficient mutant Mu50-ΔSCCmec was obtained by overexpressing Ccr recombinase, while a ccr-deleted mutant BA01611-Δccr was obtained by knocking out ccrC2 gene [13]. MRSA strain BA01611, which contains a ccrC2 gene and a type XII SCCmec element [13], was isolated in Northwest China in 2014. To produce the corresponding recombinant plasmids, the nucleotide sequences of ccrA9B3, ccrA10B1, ccrC3, ccrC4, and ccrC5 were synthesized and ligated separately into plasmid pSE1 (KpnI/EcoRI). These recombinant plasmids were then introduced into Escherichia coli (in Luria-Bertani [LB] medium suppled with 100 µg/mL ampicillin) and subsequently into related S. aureus BA01611-Δccr by electroporation (in tryptic soy broth [TSB] medium with 10 µg/mL chloramphenicol). Genomic DNA was extracted from the treated S. aureus cells using the EasyPure Genomic DNA Kit (TransGen Biotech) after they were treated with lysozyme (10 mg/mL; Solarbio) in the presence of sucrose. To detect the excision of SCCmec element from BA01611-Δccr genome, 2 primer sets (F1/R1 and F2/R2) were used to detect the attB and attSCC sites, respectively, with extracted genomic DNA as templates. The PCR protocol consisted of 95°C for 3 minutes, 95°C for 30 second, 58°C for 30 second, 72°C for 1 minute, and 4°C with 34 cycles. The resulting PCR products were sent for Sanger sequencing (Tsingke, Beijing, China).

The bacteria and plasmids used in the study are listed in Supplementary Table 4, while the primers used in the PCR assays are shown in Supplementary Table 5. These experiments allowed us to evaluate the efficacy of the novel Ccr recombinases in mediating excision of SCCmec element and provide further insights into the mechanisms of antimicrobial resistance in MRSA strains.

RESULTS

Screening Novel ccr Genes based on Nucleotide Database

To identify novel ccr genes, representative gene libraries for ccrA, ccrB, and ccrC genes were established and compared to the NCBI nucleotide total database across all genera and species. This resulted in 8574, 9838, and 1046 subject entries for ccrA, ccrB, and ccrC, respectively (Table 1 and Supplementary Table 1). After eliminating the ccr allotypes with nucleotide sequence identity ranging from 85% to 100%, 7412, 8170, and 512 sequences were left, respectively. Removing repeated results yielded 18 novel candidate ccr genes (7 ccrA, 5 ccrB, and 6 ccrC). The alignments for ccrA8 was excluded due to its low similarity (46.1%–50.2%) to all known ccrA genes (ccrA1–ccrA7). To reject false-positive results, 18 novel candidate ccr genes were aligned with 5 randomly selected allotypes of their most similar ccr genes separately. This resulted in the identification of 6 novel ccrA genes, 3 novel ccrB genes, and 3 novel ccrC genes, all of which belong to the serine recombinase family. Following the IWG-SCC committee naming guidelines, we have named these novel genes ccrA9, ccrA10, ccrA11, ccrA12, ccrA13, ccrA14, ccrB10, ccrB11, ccrB12, ccrC3, ccrC4, and ccrC5. Their nucleotide sequences are in Supplementary Material File 2.

Supplementary Table 2 shows the sequence identity range between ccrA9 and other ccrA genes, which varied from 48.5% to 83.3%, whereas ccrA10, ccrA11, ccrA12, ccrA13, and ccrA14 showed sequence identity range of 48.0%–81.3%, 48.1%–79.6%, 48.0%–81.6%, 49.7%–79.5%, and 50.1%–72.1% respectively. The ccrA genes that were most similar to the newly identified ones (ccrA9 to ccrA14) were ccrA5 (KM241), ccrA1 (GIFU12263), ccrA1 (TSU33), ccrA3 (85/2082), ccrA12 (LTH3730), and ccrA1 (ATCC15305). Similarly, the sequence identity of other types of ccrB genes ranged from 52.5% to 82.2% with ccrB10, 51.9% to 78.1% with ccrB11, and 50.1% to 82.1% with ccrB12. The most similar genes to the newly identified ccrB genes (ccrB10 to ccrB12) were ccrB1 (GIFU12263), ccrB3 (85/2082), and ccrB3 (KM241), respectively. Regarding ccrC genes, the similarity range was 68.0%–80.2% with ccrC3, 66.6%–75.8% with ccrC4, and 67.2%–80.1% with ccrC5. The most similar genes to the newly identified ccrC genes (ccrC3 to ccrC5) were ccrC1 (25/60), ccrC3 (GDK8D6P), and ccrC1 (25/60). The evolutionary relationship among the ccr genes is demonstrated in a phylogenetic tree (Figure 1), where all novel ccr genes are shown to form relatively independent clusters, indicating the uniqueness of these genes.

Novel ccr Gene Complexes and SCC/SCCmec Elements

The ccr gene complex is primarily determined by different ccr genotypes, with 9 previously reported ccr gene complexes (1–9) involved in 15 SCCmec elements (I–XV) (Figure 2). In this study, 12 novel ccr gene complexes were identified based on the genotypes of our novel ccr genes, including type 11 (ccrA9B3), type 12 (ccrA10B1), type 13 (ccrA10B10), type 14 (ccrA11B7), type 15 (ccrA11B12), type 16 (ccrA12B1), type 17 (ccrA12B3), type 18 (ccrA13B3), type 19 (ccrA14B11), type 20 (ccrC3), type 21 (ccrC4), and type 22 (ccrC5) (Table 2). Of note, there are 2 novel combinations between the ccr gene complex and the mec complex. ccrA9B3 is combined with class A mec gene complex in a M. sciuri strain GDK8D6P (Genbank accession No. CP065792.1), while ccrA10B1 is combined with class B mec gene complex in a Staphylococcus hominis strain C5 (Genbank accession No. CP093539.1) (Table 2 and Figure 2). If such combinations are found in S. aureus, they will be identified as novel SCCmec types. Except for the type 19 (ccrA14B11) and type 22 (ccrC5) ccr gene complexes, which are not located between 2 direct repeat sequences, the remaining 8 novel ccr gene complexes contribute to the formation of novel SCC elements named SCC_A9B3, SCC_A10B1, SCC_A10B10, SCC_A11B7, SCC_A11B12, SCC_A12B1, SCC_A12B3, SCC_A13B13, SCC_C3, and SCC_C4 (Supplementary Figure 1 and Table 2).

Novel Ccr Recombinase Mediate Efficient Excision of SCC Elements

The Ccr recombinase excises the SCCmec element from the bacterial genome, leading to the formation of an extrachromosomal circular intermediate (ciSCC) and the creation of 2 new att sites (attB located in the genome and attSCC located in the ciSCC). Thus, detecting the formation of attB and attSCC can determine the biological activity of Ccr recombinase. To assess the biological activity of novel Ccr recombinase encoding genes, namely ccrA9B3, ccrA10B1, ccrC3, ccrC4, and ccrC5, each group of genes was introduced separately into a mutant MRSA strain (BA01611-Δccr) lacking the ccr gene. This resulted in the successful construction of 5 recombinant strains: BA01611_Δccr-pA9B3, BA01611_Δccr-pA10B1, BA01611_Δccr-pC3, BA01611_Δccr-pC4, and BA01611_Δccr-pC5. To confirm the restoration of the S. aureus strain's ability to produce ciSSC, a mutant strain Mu50_ΔSCCmec lacking the SCCmec element was used as a positive control for the attB locus (Figure 3A) and a negative control for the attSCC locus (Figure 3B). PCR amplification targeting both the attB and attSCC loci in the wild-type MRSA strain BA01611 yielded positive results, while those in the mutant strain BA01611_Δccr were negative, indicating that the lack of Ccr recombinase resulted in the inability of BA016111_Δccr to excise the SCCmec element. However, after introducing ccrA9B3, ccrA10B1, ccrC3, ccrC4, and ccrC5 into BA01611_Δccr, respectively, positive results were obtained for PCR amplification of the attB and attSCC loci (Figure 3). These results demonstrate that overexpression of these Ccr recombinases in BA01611_Δccr restored the ability of this S. aureus strain to produce ciSCC, indicating that the 5 groups of Ccr recombinases are biologically active.

Distribution of Novel ccr Genes

We investigated the distribution of the novel ccr genes in various staphylococcal isolates. Supplementary Table 3 shows that a total of 13 staphylococci isolates encoding ccrA9 were identified from China (5 M. sciuri and 1 S. lentus), Germany (3 S. aureus and 2 S. carnosus), France (1 S. lugdunensis), and Korea (1 S. saprophyticus). Similarly, a total of 9 staphylococci isolates encoding ccrA10 were identified from Germany (1 S. xylosus, 1 S. vitulinus, 1 S. condimenti, and 1 S. carnosus), the United States (1 S. hominis and 1 S. saprophyticus), Korea (2 S. nepalensis), and China (1 S. xylosus). We also found 3 staphylococci isolates encoding ccrA11 from China (2 S. delphini) and Brazil (1 S. chromogenes). Additionally, 3 staphylococci isolates encoding ccrA12 gene were identified from Germany (1 S. xylosus), Korea (1 S. chromogenes), and Thailand (1 S. succinus), while 1 Staphylococcus (M. sciuri) isolate encoding ccrA13 was isolated from China. Moreover, 1 Staphylococcus isolate encoding ccrA14 gene was isolated from the Czech Republic (S. simiae). Three staphylococci isolates encoding ccrB10 were identified from China (1 S. xylosus), the United States (1 S. saprophyticus), and Germany (1 S. vitulinus). Similarly, 1 Staphylococcus isolate encoding ccrB11 was identified from the Czech Republic (1 S. simiae), while 15 staphylococci isolates encoding ccrB12 were identified from Germany (9 S. aureus), China (2 S. delphini and 1 S. haemolyticus), Korea (1 S. haemolyticus), Brazil (1 S. chromogenes), and the United Kingdom (1 S. caeli). Finally, we found 4 staphylococci isolates encoding ccrC3 from China (2 M. sciuri), the United States (1 S. cohnii), and the United Kingdom (1 S. cohnii), 2 isolates encoding ccrC4 gene from China (1 S. lentus) and Denmark (1 S. vitulinus), and 1 isolate encoding the ccrC5 from China (1 S. cohnii).

DISCUSSION

Bacterial molecular typing is a crucial aspect of epidemiological surveillance and investigation, providing insight into the epidemiological connection within outbreak environments. As the cost of whole-genome sequencing (WGS) continues to decrease, WGS-based methods are expected to become the gold standard for bacterial molecular typing [14]. It includes several approaches such as single-nucleotide polymorphism-based, core genome alignment, gene-by-gene comparison, and accessory genome typing [15]. With the advent of massive and detailed bacterial genome sequences, a new opportunity has emerged to search for novel SCCmec using genome databases. In the present study, 12 novel ccr genes were identified by searching the staphylococcal genome database. These genes were combined into 12 novel ccr gene complexes. Among them, 2 novel combinations between the ccr and the mec complex were identified. ccrA9B3 is combined with class A mec gene complex, and ccrA10B1 is combined with class B mec gene complex. The novel combinations will only be identified as novel SCCmec types when found in S. aureus that can need classification for further assessments.

SCCmec typing is a rapid and highly effective method used for typing MRSA isolates, aiding in the comprehensive investigation of clonal transmission and enhancing our understanding of MRSA's changing epidemiology. Since the description of SCCmec type I in 2001 in an MRSA strain NCTC10442, 15 SCCmec types have been identified [2, 16], with only 4 having been reported in the last decade (XII–XV) [10, 16–18.] Ccr recombinases play a key role in excising and integrating SCC/SCCmec elements to form a ciSCC, facilitating transfer among staphylococci through transduction or natural transformation in the biofilm [9, 19, 20]. Increasing the expression level of Ccr recombinase significantly boosts the frequency of SCCmec excision [13, 21], and antimicrobial agents targeting DNA replication and repair or UV radiation can promote SCCmec transfer by triggering a bacterial SOS response and upregulating the expression of ccr gene [22, 23].

The intricate evolution of SCCmec elements entails a multifaceted process, with early involvement of M. sciuri particularly emphasized in the pioneering stages [24, 25]. The putative evolutionary precursor of mecA, identified as mecA1, emerges as a gene ubiquitously present in M. sciuri [24]. Noteworthy contributions to the assembly of the mec complex are attributed to S. vitulinus and S. fleurettii [25]. Additionally, findings from another study suggest that M. sciuri may serve as the donor of the ccr complex crucial for the assembly of SCCmec [26]. This investigation unveiled novel ccr gene complexes within the genomic landscape of M. sciuri strains, underscoring its pivotal role in shaping the evolution of both ccr gene complexes and SCCmec elements. Analysis of the postulated evolutionary route suggested that the SCCmec elements were assembled from genetic material from various bacterial species in different habitats of different countries, and the hsdS restriction-modification profile may facilitate the genetic exchange of SCCmec elements among different staphylococcal species [5].

In accordance with the IWG-SCC criteria, phylogenetically distinct ccr genes have been determined with less than 50% DNA sequence similarity to each other, while the nucleotide similarity of the ccr gene subtypes is more than 85% [1]. Eight ccrA, 9 ccrB, and 2 ccrC have been reported, forming 10 ccr gene complexes designated as type 1 (ccrA1B1), type 2 (ccrA2B2). type 3 (ccrA3B3), type 4 (ccrA4B4), type 5 (ccrC1), type 6 (ccrA5B3), type 7 (ccrA1B6), type 8 (ccrA1B3), type 9 (ccrC2), and type 10 (ccrA8B9). The 12 novel ccr genes and 12 novel ccr gene complexes identified here will greatly enrich the ccr gene pool. The newly named ccrC5 gene was originally designated as ccrC2 gene, which was first reported in 2010 [27]. With the agreement of the IWG-SCC committee, the ccrC2 gene was renamed as ccrC5 to avoid confusion with the well-known ccrC2 gene discovered in 2015 [10]. Additionally, the type 6 (ccrA5B3) and type 10 (ccrA8B9) ccr gene complexes have not been found to be involved in construction of SCCmec elements [6, 27].

To avoid omissions and inaccuracies in research findings, the strength of data sharing and development of algorithms should be underlined. This study gathers the total diversity of ccr genes and SCC/SCCmec elements. With the release and in-depth analysis of more genomic diversities of all species except for S. aureus from different reservoirs, the evolutionary pathway of important gene islands such as SCCmec will be more clearly elucidated in the future.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. J. H. and J. X. conducted experiments and analyzed the data. X. W., X. X., Y. M., Z. Z., L. Z., and Z. M. provided technical support. X. Z., A. L., and H. X. designed the experiment and supervised the project. J. H. and H. X. wrote the manuscript. P. L., X. Z., A. L., and H. X. revised the manuscript. All authors have reviewed the manuscript.

Acknowledgment. The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn/) for the expert linguistic services provided.

Financial support. This work was supported by the Shaanxi Fundamental Science Research Project for Chemistry and Biology (grant number 22JHQ055); and the National Natural Science Foundation of China (grant number 31972652).

References

Author notes