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. 2017 Feb 23;61(3):e02500-16. doi: 10.1128/AAC.02500-16

Full-Genome Sequencing Identifies in the Genetic Background Several Determinants That Modulate the Resistance Phenotype in Methicillin-Resistant Staphylococcus aureus Strains Carrying the Novel mecC Gene

Catarina Milheiriço a,✉, Hermínia de Lencastre a,b, Alexander Tomasz b
PMCID: PMC5328527  PMID: 28069659

ABSTRACT

Most methicillin-resistant Staphylococcus aureus (MRSA) strains are resistant to beta-lactam antibiotics due to the presence of the mecA gene, encoding an extra penicillin-binding protein (PBP2A) that has low affinity for virtually all beta-lactam antibiotics. Recently, a new resistance determinant—the mecC gene—was identified in S. aureus isolates recovered from humans and dairy cattle. Although having typically low MICs to beta-lactam antibiotics, MRSA strains with the mecC determinant are also capable of expressing high levels of oxacillin resistance when in an optimal genetic background. In order to test the impact of extensive beta-lactam selection on the emergence of mecC-carrying strains with high levels of antibiotic resistance, we exposed the prototype mecC-carrying MRSA strain, LGA251, to increasing concentrations of oxacillin. LGA251 was able to rapidly adapt to high concentrations of oxacillin in growth medium. In such laboratory mutants with increased levels of oxacillin resistance, we identified mutations in genes with no relationship to the mecC regulatory system, indicating that the genetic background plays an important role in the establishment of the levels of oxacillin resistance. Our data also indicate that the stringent stress response plays a critical role in the beta-lactam antibiotic resistance phenotype of MRSA strains carrying the mecC determinant.

KEYWORDS: MRSA, mecC, beta-lactams, resistance

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multiresistant human pathogens worldwide, causing infections in hospitals, in the community, and in livestock (1). Most MRSA strains are resistant to beta-lactam antibiotics since they carry the central element of methicillin resistance: the mecA gene, which encodes an extra penicillin-binding protein (PBP2A), an enzyme involved in the synthesis of cell wall peptidoglycan. PBP2A has low affinity for virtually all beta-lactam antibiotics, allowing it to continue catalyzing cell wall synthesis even in the presence of beta-lactams in the medium (2). The mecA gene resides on a mobile genetic element of heterologous origin, the staphylococcal cassette chromosome mec (SCCmec). SCCmec elements are characterized by the presence of two essential loci: the mec gene complex, comprising the methicillin resistance determinant with intact or truncated copies of the mec regulatory genes (mecI, mecR1, and mecR2) and the ccr gene complex, which encodes site- and orientation-specific recombinases responsible for SCCmec mobilization (3). While extensive diversity has been reported in the genetic organization of SCCmec elements, virtually no variation has been identified in the sequence of the mecA determinant in MRSA strains (4).

In 2011, a new methicillin resistance determinant, the mecC gene, was identified in S. aureus isolates recovered from humans and dairy cattle. The mecC determinant was able to produce low-level resistance to beta-lactam antibiotics such as cefoxitin and oxacillin (5, 6). Following its first description, several studies reported the occurrence of mecC-carrying MRSA strains in a broad range of host species, including humans, livestock, companion animals, and wildlife (1). The frequency of MRSA isolates carrying the mecC determinant reported so far is relatively low. However, due to the lack of longitudinal epidemiological studies tracking the dissemination of mecC-carrying MRSA strains, it is difficult to ensure the true distribution and prevalence of these strains. The mecC determinant shares only 69% nucleotide identity with mecA gene from MRSA strain N315 (4) and is integrated in a new SCCmec structure (SCCmec XI) that also contains homologs of the mecA regulatory genes, the blaZ gene, and genes for site-specific recombinases, ccrA and ccrB (5).

Recent studies demonstrated that besides having a different nucleotide content and distinct biochemical properties from the gene and protein encoded by the mecA gene of MRSA strain COL, the protein encoded by mecC (PBP2ALGA) is able to deliver high and homogeneous levels of oxacillin resistance when expressed free of its regulatory elements and in an appropriate genetic background (7). However, it has been recently proved that PBP2ALGA does not mediate resistance to penicillin and in order to express broad-spectrum resistance to beta-lactams antibiotics in its natural genetic background, it requires the expression of the beta-lactamase gene (blaZLGA251) located adjacent to mecC on the SCCmec type XI element (8).

Due to the typically low MICs of oxacillin and cefoxitin, the absence of mecA gene, and the lack of availability of efficient, quick tests to identify the new PBP2A homolog (5, 6), strains carrying mecC could be potentially misidentified as methicillin-susceptible S. aureus (MSSA), which eventually may generate highly resistant strains if treated with beta-lactam antibiotics.

This new resistance determinant in an SCCmec of small size (30 kb)—SCCmec type XI—is likely to be more transferable between strains. This possibility in conjunction with its ability to cause a wide variety of infections (including severe and fatal infections) in a number of host species, including humans (9–13), and its capacity to deliver high levels of oxacillin resistance when expressed in an optimal genetic background (7) constitute matters of concern.

MRSA strains with the mecC determinant have typically low MICs for beta-lactam antibiotics. No data are available on the impact of extensive beta-lactam selection on the possible emergence of mecC-carrying strains with high levels of antibiotic resistance. One of the purposes of the studies described here was to test this.

RESULTS

Generation of LGA251 mutants with increased levels of oxacillin resistance.

In order to evaluate the impact of extensive beta-lactam treatment on the selection of mecC-carrying strains with high levels of oxacillin resistance, we exposed the mecC MRSA prototype strain, LGA251, to step selection using increasing concentrations of oxacillin in the growth medium. Briefly, we started by picking a colony of LGA251 from a tryptic soy agar (TSA) plate containing 12.5 μg/ml of oxacillin. This colony was then used as the inoculum to generate an overnight culture on tryptic soy broth (TSB) containing 12.5 μg/ml of oxacillin. After a few serial passages in TSB with increasing concentrations of oxacillin in the medium, we recovered LGA251 derivatives—LGA251-50, LGA251-100, and LGA251-800—that were able to grow in the presence of 50, 100, or 800 μg/ml of oxacillin, respectively. In contrast to the parental strain, LGA251, these LGA251 mutants showed homogeneous oxacillin resistance when tested by population analysis (Fig. 1).

FIG 1.

FIG 1

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Antibiotic resistance profile of S. aureus strain LGA251 and its derivatives—LGA251-50, LGA251-100, and LGA251-800—with increased levels of oxacillin resistance. Antibiotic resistance was determined by population analysis. CFU were calculated by counting colonies after 48 h of incubation on tryptic soy agar plates containing increasing concentrations of oxacillin.

The highly oxacillin-resistant mutant LGA251-800 carries a point mutation in the TPase domain of mecC.

Sanger sequencing of the entire mec region and the determinants of PBP2 and -4 was carried out in order to identify if any of these regions could be implicated in the mechanism of increased resistance of the three LGA251 derivative strains recovered from the step selection procedure. No mutations were found in the mecC regulatory genes or in the determinants of PBP2 and PBP4 (Table 1). However, a point mutation was identified in the mecC gene of the most resistant derivative, LGA251-800. This was a point mutation in the transpeptidase (TPase) domain of the mecC gene (located 1,327 bp from the mecC start codon) (Table 1), which led to a substitution in the 443rd amino acid of the product of mecC. The product of wild-type mecC from LGA251 has a phenylalanine and that from LGA251-800 has a leucine (both nonpolar hydrophobic amino acids) at this position.

TABLE 1.

Loci screened by Sanger sequencing for the presence of mutations in LGA251 derivative strains

Strain Locus
mecC mecC regulatory region PBP2 PBP4
LGA251 WTa WT WT WT
LGA251-50 WT WT WT WT
LGA251-100 WT WT WT WT
LGA251-800 WT T1327C (F443L) WT WT
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a

WT, wild-type allele.

Search for additional mutations in oxacillin-resistant mutants of LGA251.

Whole-genome sequencing (WGS) was used to search for possible additional genetic determinants that were affected during the step selection procedure.

Eleven single nucleotide polymorphisms (SNPs) affecting eight open reading frames (ORFs) were identified (Fig. 2 and 3; see also Table S3 in the supplemental material). As expected, accumulation of mutations occurred during selection: the first derivative strain (LGA251-50) that was able to grow in the presence of 50 μg/ml of oxacillin in the growth medium was the one with the fewest mutations, and these mutations were maintained in the more resistant derivative strains (LGA251-100 and LGA251-800). In addition, two genetic regions, comprising 10 different ORFs, were deleted during the step selection process (Fig. 3 and Table S3): one at the beginning of the step selection procedure (detected in the three LGA251 derivative strains) and the other only in the final steps of the step selection in the most resistant LGA251 derivative, LGA251-800.

FIG 2.

FIG 2

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Overview of chromosomal regions affected during the step selection procedure. The presence of mutations across the genomes of each of the LGA251 derivative strains is shown as red bars.

FIG 3.

FIG 3

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Summary of the results obtained in the WGS analysis of LGA251 derivatives. The presence of mutations is highlighted in blue. Highlighted in red are genes that may be involved in the increased levels of oxacillin resistance of the LGA251 derivatives.

ORFs containing single nucleotide polymorphisms were scattered throughout the core genome of LGA251, and they affected several metabolic functions (Fig. 2 and 3). Besides the mutation in mecC (mecC*), which was detected in the most resistant derivative strain (LGA251-800), no other genes linked to the mecC-encoded resistance mechanism were detected.

Impact of mecC* mutation on antibiotic resistance in two different genetic backgrounds.

In order to test the impact of the mutated form of the mecC gene (mecC*) on the beta-lactam resistance level, we overexpressed the mecC* gene in the backgrounds of the susceptible strains RN450 and COL-S. The mutant strain RN450+pBCB8::mecC* showed only a modest increase in the oxacillin MIC compared to that of the parental strain, RN450, by Etest (0.19 μg/ml and 0.38 to 0.5 μg/ml, respectively). On the other hand, the mutant strain COL-S+pBCB8::mecC* showed a massive increase in the oxacillin MIC (0.38 μg/ml up to >256 μg/ml) compared to that of the parental strain, COL-S. Population analysis profiles confirmed these results: the oxacillin MIC for mutant strain RN450+pBCB8::mecC* was 0.75 μg/ml, and the oxacillin MIC for the parental strain, RN450, was 0.1 μg/ml. In contrast, the oxacillin MIC of mutant strain COL-S+pBCB8::mecC* was 800 μg/ml, and the oxacillin MIC for the parental strain, COL-S, was 0.75 μg/ml (Fig. 4).

FIG 4.

FIG 4

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Effect of the introduction of plasmid-borne copies of mecC* on the oxacillin resistance profiles of strains RN450 (A) and COL-S (B). Antibiotic resistance was determined by population analysis. CFU were counted after 48 h of incubation on tryptic soy agar containing increasing concentrations of oxacillin.

These results show that the mutated form of the mecC gene found in LGA251-800 was able to deliver high levels of oxacillin resistance in an optimal genetic background (COL-S). In previous studies, we have already shown that the wild-type form of mecC can produce high levels of oxacillin resistance in the COL-S background (7). However, in contrast to the results obtained in this study with mecC* (the mutant form of mecC) (Fig. 4A), the moderate and heterogeneous degree of oxacillin resistance delivered by the wild-type form of mecC when expressed in RN450 genetic background or in its natural genetic background (LGA251) could be boosted to high and homogeneous resistance by growing the bacteria under conditions of stringent stress (7) induced by the addition of subinhibitory concentrations of mupirocin to the growth medium (Fig. 5).

FIG 5.

FIG 5

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Effect of mupirocin on the oxacillin resistance profiles of strains LGA251 (A) and RN450mecC (B). Antibiotic resistance was determined by population analysis. CFU were counted after 48 h of incubation on tryptic soy agar containing increasing concentrations of oxacillin.

Genes contributing to the level of oxacillin resistance in derivatives of LGA251.

Surprisingly, and in spite of their increased levels of oxacillin resistance, no genes with a direct link to the methicillin resistance mechanism were detected in the WGS data reported here for strains LGA251-50 and LGA251-100. These two strains differ only by an SNP mutation in LGA251-100, in a gene that belongs to the ABC transport system. Therefore, this gene appears to be associated with the increased MIC of oxacillin for strain LGA251-100. Cloning studies are under way to confirm this.

Regarding the genes affected in LGA251-50, we were not able to identify any obvious candidate gene responsible for the increased level of oxacillin resistance. However, among the altered genes, we identified one involved in guanine metabolism that may be involved in the response to stress conditions. Since the involvement of the stringent stress in the beta-lactam resistance phenotype of MRSA strains has recently been demonstrated (14), this gene may be one candidate responsible for the oxacillin resistance levels observed.

DISCUSSION

Most methicillin-resistant S. aureus (MRSA) strains carry the chromosomal mecA determinant, which equips them with resistance against the large class of beta-lactam antibiotics. The emergence of MRSA represents a major public health problem worldwide. Multiresistant and epidemic strains of MRSA may carry many virulence traits of S. aureus and also have the capacity to develop resistance to several antimicrobial agents. The first MRSA strains appeared in 1960, soon after the introduction of methicillin into clinical practice, and since then, MRSA lineages have spread globally, causing infections both in humans and in animals. The first reports on a new methicillin resistance genetic determinant, mecC, appeared in 2011 (5, 6). The mecC gene is carried on a mobile genetic element of small size (SCCmec XI). Its capacity to deliver high levels of oxacillin resistance when expressed in an optimal genetic background (7) and the ability of mecC-carrying strains to cause a wide variety of infections in several host species have raised concern.

We exposed the prototype mecC-carrying MRSA strain, LGA251, to increasing concentrations of oxacillin and monitored the appearance of mutations in derivative strains by WGS in order to assess the impact of extensive beta-lactam selection on the possible emergence of mecC-carrying strains with high levels of antibiotic resistance. We have seen that LGA251 could easily adapt to high concentrations of oxacillin in its growth medium. Several mutations have occurred over time. The first mutations occurred in genes belonging to the genetic background, but nevertheless, they were responsible for moderately increased levels of oxacillin resistance. This was not surprising, since several studies involving transposon mutagenesis have previously identified a number of auxiliary genes in the bacterial genetic background that influence the beta-lactam resistance phenotype in mecA-carrying MRSA strains (15–18). However, to the best of our knowledge, none of the genes identified in the studies described here were previously identified as being involved in the establishment of beta-lactam resistance levels in mecA-carrying MRSA strains. While clearly essential for oxacillin resistance (19), the presence of the mecC genetic determinant alone could not explain the difference in oxacillin resistance levels when expressed in different genetic backgrounds.

A mutation affecting mecC was detected only in the most resistant derivative strain, LGA251-800. Despite its being mutated, we have seen that mecC* is still functional and able to deliver high levels of oxacillin resistance when expressed in an optimal genetic background (Fig. 4B). However, we cannot ascertain that this mutation alone was responsible for the high level of oxacillin resistance found in LGA251-800. The level of resistance in strains with mecC* is dependent on the genetic background (Fig. 4). Moreover, in a previous study (7), we have shown that the same phenomenon—delivery of high-level resistance—also happens with the wild-type copy of mecC when expressed in the optimal genetic background of strain COL-S. The COL-S genetic background was used to ascertain the functionality of mecC* since it is readily manipulatable and was previously seen to be appropriate for the expression of high levels of oxacillin resistance of the wild-type copy of mecC (7). However, in order to prove the efficacy of the mecC* mutation in terms of the oxacillin resistance phenotype of LGA251-800, more studies involving the replacement of the wild-type copy of mecC by the mutated form mecC* in its natural genetic background (ST425) are still needed. Interestingly, another recent study has also identified a mutation in the mecC gene causing a Val546Ile substitution in the transpeptidase domain of PBP2ALGA; however, the identified mutation was responsible for decreased levels of cefoxitin resistance, demonstrating the importance of the valine residue in position 546 for specifically mediating cefoxitin resistance in PBP2ALGA (8). In contrast, the mutation described here and found in mecC* of LGA251-800 is supposedly responsible for increased levels of resistance to oxacillin.

Recent studies (14, 20, 21) showed that under stringent stress conditions, mecA transcription and translation are increased despite the low growth rate of the MRSA strains tested, clearly showing that the stringent stress response plays a critical role in the level of beta-lactam resistance of MRSA strains. Our results indicate that similarly to what happens with mecA, expression of resistance in strains carrying mecC is also stimulated by induction of the stringent stress response (Fig. 5). However, mecC*, the mutated form of the mecC gene found in LGA251-800, appears to be exempt from the stringent control (Fig. 4).

The observations described in this study clearly show that reliable detection and monitoring of mecC MRSA strains with appropriate phenotypic and molecular screening methods are needed, as these strains are able to rapidly adapt to antibiotic pressure. Moreover, our results indicate that key determinants of the oxacillin resistance level reside in the genetic background of the S. aureus carrying the mecC genetic determinant, fully confirming the conclusions reached in studies of similar design in which the genetic determinant tested was the mecA gene (14, 20, 21). The striking increase in oxacillin resistance caused by induction of the stringent stress response (mupirocin) provides yet another line of evidence for the basic similarities in the mechanism of beta-lactam antibiotic resistance in MRSA strains carrying either the mecA or the mecC determinant.

MATERIALS AND METHODS

Bacterial strains, media, and growth conditions.

The characteristics of bacterial strains and plasmids used in the present study are listed in Table S1. S. aureus strains were grown on tryptic soy broth (Difco Laboratories) or on tryptic soy agar (Difco Laboratories) at 37°C with aeration. Escherichia coli strains were grown in Luria-Bertani broth (Difco Laboratories) or on Luria-Bertani agar (Difco Laboratories) with aeration at 37°C. Recombinant E. coli strains were selected and maintained with ampicillin at 100 μg/ml. Recombinant S. aureus strains were selected and maintained in a medium supplemented with the appropriate antibiotics (neomycin at 50 μg/ml and kanamycin at 50 μg/ml). CdCl2 (0.2 M) was added to the medium to induce the transcription of mecC and the mutated form of mecC, mecC*, in recombinant S. aureus strains.

Step selection procedure.

A colony of the mecC-carrying MRSA prototype strain LGA251 was picked from a TSA plate containing 12.5 μg/ml of oxacillin. This colony was then used as the inoculum of an overnight culture in TSB medium that was serially passaged with increasing concentrations of oxacillin. The oxacillin concentration was doubled in each passage.

Antibiotic susceptibility.

Antibiotic susceptibility was determined by Etest (bioMérieux) and population analysis profiles (PAPs). The Etest was done by spreading a small aliquot of overnight culture diluted to an optical density at 620 nm (OD620) of 0.08 onto TSA plates and adding an oxacillin Etest strip onto the surface of the plates (20). PAP testing was done on overnight cultures diluted with TSB and plated onto TSA plates with or without a sub-MIC of mupirocin (0.03 mg/liter) (20) and serial (2-fold) dilutions of oxacillin according to the population analysis method described previously (22). MICs of oxacillin were determined after 48 h of incubation at 37°C.

DNA manipulation.

Total DNA from S. aureus was isolated from bacterial cultures with the Wizard genomic DNA (gDNA) purification kit (Promega) according to the manufacturer's recommendations and using lysostaphin (0.5 mg/ml) and RNase (0.3 mg/ml) in the lysis step. Plasmid DNA was isolated from bacterial cultures with the High Pure plasmid isolation kit (Roche). For plasmid DNA isolation from S. aureus strains, the culture pellets were resuspended in suspension buffer supplemented with 0.1 mg/ml of lysostaphin and incubated at 37°C for 60 min. Restriction enzymes were used as recommended by the manufacturer (New England BioLabs). Routine PCR was performed with GoTaq Flexi DNA polymerase (Promega). PCR amplification of cloning inserts was performed by high-fidelity PCR (Pfu Turbo DNA polymerase; Stratagene). DNA purification from PCR and digestion reactions was performed with a High Pure PCR product purification kit (Roche). For ligation protocols, the inserts and linearized plasmids were resolved in a low-melting-point agarose gel (1%) (Invitrogen) and DNA bands were purified with a High Pure PCR product purification kit (Roche) by following the manufacturer's recommendations. DNA Sanger sequencing was performed by Macrogen. All primers used in this study are listed in Table S2.

Whole-genome sequencing.

Genomic DNA (gDNA) of derivatives LGA251-50, LGA251-100, and LGA251-800 was extracted using the phenol-chloroform method (23). Whole-genome sequencing was performed using the Illumina HiSeq 2500 platform (Rockefeller University Genomics Resource Center). The genome analysis was done by mapping the single-end reads of each of the derivatives against the LGA251 genome (available at NCBI under accession number NC_017349.1) and by making an SNP calling using the CLC Genomics Workbench software (CLCbio; Qiagen). In order to discard sequencing or assembly errors, mutations present in the LGA251 derivatives were confirmed by Sanger sequencing (Macrogen).

Introduction of the mecC and mecC* genes into S. aureus strains RN450 and COL-S.

The mecC* gene (a mutated form of the mecC gene identified in lab construct LGA251-800; see Results) was cloned into a shuttle vector, pBCB8, equipped with a cadmium-inducible promoter. The gene was amplified by PCR using the primers mecALGAF5 and mecALGAR6 from LGA251 (7). The product was ligated into the SacI and EcoRI sites of the pBCB8 plasmid. The recombinant plasmid was then introduced into E. coli DH5α (24) and was named pBCB8::mecC*. The nucleotide sequence was confirmed by Macrogen. Next, the recombinant plasmid pBCB8::mecC* was introduced into the restriction-deficient S. aureus strain RN4220 (25) by electroporation (26), followed by transduction (24) using phage 80α into the oxacillin-susceptible S. aureus strain RN450 and into COL-S to produce strains RN450mecC* and COL-SmecC*, respectively. The recombinant plasmid pBCB8::mecC (7) was also introduced through transduction in the genetic background of RN450. In the transductants, transcription of the mecC and mecC* genes was under the control of the cadmium-inducible promoter.

Accession number(s).

Data for the genome sequencing samples have been deposited in the European Nucleotide Archive (ENA) under sample numbers ERS1486578 (LGA251-50), ERS1486580 (LGA251-100), and ERS1486582 (LGA251-800).

Supplementary Material

Supplemental material
supp_61_3_e02500-16__index.html (1.1KB, html)

ACKNOWLEDGMENTS

We thank Susana Gardete for providing technical assistance in the handling of the genomic DNA.

This work was financially supported by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI), by national funds through Fundação para a Ciência e a Tecnologia (FCT), and by U.S. Public Health Service award 2 R01 AI457838-15 to A. Tomasz. C.M. was supported by grants SFRH/BPD/63992/2009 and SFRH/BPD/111697/2015 from FCT.

We have no conflicts of interest to declare.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02500-16.

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