MLS B -Resistant Staphylococcus aureus in Central Greece: Rate of Resistance and Molecular Characterization

Bacterial isolates and susceptibility testing

A total of 2,893 consecutive S. aureus isolates recovered from clinical samples, including bloodstream infections, wounds, pneumonia, arthritis, soft tissue, and skin infections of individual patients (hospitalized and outpatients) admitted in the University Hospital of Larissa (UHL) between 2012 and 2017, were included in the study. UHL is a 800-bed tertiary care hospital in Central Greece, serving a population of 1,000,000 habitants. Duplicate isolates from the same patients, even if the site of infection was different, were excluded. Identification to species level and susceptibility testing against various antimicrobial agents were performed by the automated system Vitek^®2 (BioMerieux, France). Minimum inhibitory concentrations (MICs) were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria (www.eucast.org). To confirm the inducible MLS_B (iMLS_B) phenotype, double-disk diffusion test was performed in all isolates that expressed resistance to erythromycin (MICs >2 mg/L) and susceptibility to clindamycin (MICs >0.5 mg/L). ¹³

Two hundred seventy-five MLS_B-resistant S. aureus were selected to be further studied. These isolates were selected as representatives of different MLS_B phenotype (inducible or constitutive or others), MRSA or methicillin-sensitive S. aureus (MSSA) phenotypes, susceptibility pattern to various antimicrobial agents, site of infection, age of patient, time of isolation, and ward of admission.

Detection of resistance determinants

Genes encoding for resistance to MLS_B were detected by polymerase chain reaction (PCR), using specific primers for ermA, ermB, ermC, ermF, ermY, ermT, msrA, msrB, ereA, ereB, lnuA, lnuB, and mphC. ¹⁴ Both strands of the PCR products were sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA).

Multilocus sequence typing of S. aureus isolates

The isolates were typed by Multilocus Sequence Typing (MLST). MLST was performed by sequencing of the seven gene loci, arcC, aroE, glpF, gmk, pta, tpi, and yqi. ¹⁵ The data for S. aureus alleles and sequence types (STs) were obtained through the MLST database at https://pubmlst.org/saureus. The eBurst v.3 was used to clarify the related STs into clonal complexes (CCs). ¹⁶

Illumina sequencing

Eight erm-positive staphylococcal isolates were selected for WGS. These isolates were selected as representative of different STs, isolation periods, and origin.

Genomic DNAs from the clinical isolates were extracted using the DNASorb-B Kit (Sacace Biotechnologies Srl, Como, Italy). Multiplexed DNA libraries were prepared, using the Nextera XT Library Preparation Kit, and 300-bp paired-end sequencing was performed on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA) using the MiSeq v3 600-cycle Reagent Kit. Initial paired-end reads were quality trimmed, using the Trimmomatic tool v0.33, ¹⁷ with the sliding window size of 4 bp, required average base quality ≥17, and minimum read length of 48 bases. Then, reads were assembled by use of the de Bruijn graph-based de novo assembler SPAdes v3.9.1, ¹⁸ using k-mer sizes 21, 33, 55, 77, 99, and 127. For sequence analysis and annotation, the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST), the ISfinder database (www-is.biotoul.fr/), and the open reading frame finder tool (www.bioinformatics.org/sms) were utilized. Comparative genome alignments were performed using the Mauve v2.3.1 program. ¹⁹

Detection of erm-carrying mobile elements

Based on the results from Illumina sequencing (see erm-associated mobile elements subhead under the Results section), eight PCRs targeting characteristic regions of erm-carrying mobile elements sequenced during this study were designed. The selected regions included: (i) the ermA-carrying transposon Tn554, (ii) the ermA-carrying transposon Tn6133, (iii) and the ermC-carrying plasmid pSau-2716Lar. All erm-positive clinical strains were screened for the presence of the regions described above by the use of specific primers (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr). PCR products were sequenced as described above.

Statistical analysis

For analyzing the correlation between year when the samples were collected, and the percentage of MLS_B-resistant S. aureus, we used the method Pearson's correlation. For analyzing the possible difference between the percentage of MLS_B-resistant MSSA and MLS_B-resistant MRSA, we used the Mann–Whitney U test. In both tests, values of p < 0.05 were considered as statistically significant. The computation was done by the program Review Manager (RevMan) Version 5.3. ²⁰

Nucleotide sequence accession numbers

The nucleotide sequences of the erm-carrying fragments described in this study have been deposited in GenBank under accession numbers MH423311, MH423312, and MH454238-MH4543. Whole-genome assemblies of S. aureus isolates were deposited in NCBI under accession number PRJNA487708.

MLS_B-R staphylococci

Of the 2,893 S. aureus, 1,161 isolates (40.2%) exhibited resistance to at least one of the MLS_B agents (Table 1). The majority of MLS_B-resistant S. aureus (1,077 isolates) exhibited resistance to erythromycin and clindamycin, and expressed either the consecutive MLS_B-phenotype (cMLS_B) or the inducible MLS_B-phenotype (iMLS_B) (765 and 312 isolates, respectively). Among the remaining MLS_B-resistant isolates, 84 were only resistant to erythromycin (MS_B phenotype). No isolate with resistance to clindamycin (L phenotype) was detected. Table 1 describes the rate of MLS_B-resistance and the distribution of phenotypes among MRSA and MSSA during the 6-year study period; the rate of MLS_B-resistance was statistically significantly higher in MRSA than in MSSA (p = 0.002). Additionally, there was a statistically significant positive correlation between the isolation year and the percentage of MLS_B-resistant S. aureus (Correlation Coefficient = 0.839, and p = 0.037).

In addition, according to the susceptibility results, 53.8% of the MLS_B-resistant S. aureus also exhibited resistance to levofloxacin, 52.0% to tobramycin, 32.4% to tetracycline, 28.7% to fusidic acid, 9.5% to gentamicin, 4.4% to trimethoprim–sulfamethoxazole, and 2.2% to rifampicin. None of the MLS_B-resistant isolates expressed resistance to mupirocin.

To determine the resistance mechanisms, 275 MLS_B-resistant S. aureus, including 81 MSSA and 194 MRSA isolates, were selected to be further studied. Among the MSSA isolates (n = 81), 22 expressed the cMLS_B phenotype, 42 expressed the iMLS_B phenotype, and 17 the MS_B phenotype (Table 2). Most of MSSA isolates carried the ermC gene (n = 38), whereas 26 isolates were positive for the ermA. The 17 MSSA isolates expressing the MS_B phenotype carried the msrA gene. These data are in agreement with the findings of previous studies showing that ermC gene was common among MSSA as well as coagulase-negative staphylococci.^21,22

Moreover, 147 of the MRSA isolates expressed the cMLS_B phenotype, whereas 44 MRSA expressed the iMLS_B phenotype (Table 3). The three remaining MRSA expressed the MS_B phenotype and carried the msrA gene. The ermA gene was identified in the majority of MRSA (n = 153), of which 133 isolates expressed the cMLS_B phenotype. Thirty-seven MRSA isolates, expressing either the cMLS_B (n = 13) or the iMLS_B (n = 24) phenotypes carried the ermC gene. One MRSA isolate was positive for two genes, ermA and ermC, encoding erythromycin resistance. The later genotype has been rarely reported.^13,21

Population structure of erm-positive S. aureus isolates

The population structure of the 275 MLS_B-resistant S. aureus isolates is shown in Tables 2 and 3. The majority of MLS_B-resistant MRSA and MSSA belonged to the CC5. The group of 81 MSSA isolates was distributed to 13 STs, with STs 121 (n = 17), 145 (n = 10) and 282 (n = 10) accounting for 37 of the isolates. Most of ST282 isolates were associated with ermA, whereas most of the isolates belonging to ST121 carried the ermC gene. All ST145 MSSA isolates included the msrA gene. On the other hand, less genetic diversity was found in the group of MRSA isolates. Twelve different STs were identified among the group of the 194 MRSA isolates. ST225, which belongs to CC5, was the most prevalent, accounting for 137 MRSA isolates. The majority of ST225 MRSA isolates were associated with the presence of ermA. MRSA attributed to ST225 are predominant among isolates recovered from hospitals in European countries.^23,24 Thirty-one out of 37 ermC-carrying MRSA isolates were distributed in STs 1 (n = 11), 80 (n = 10), 239 (n = 6), and 728 (n = 4).

erm-associated mobile elements

To define the regions flanking the erm resistance genes, the genome of staphylococcal isolate representatives of different erm genes, sequencing types (STs), and origin was completely sequenced.

De novo assembly and analysis of sequencing data showed that, in five out of six sequenced ermA-positive S. aureus isolates (ST5 S. aureus Sau-2933Lar, ST7 S. aureus Sau-3805Lar, ST30 S. aureus Sau-3298Lar, ST225 S. aureus Sau-3323Lar, and ST225 S. aureus Sau-3535Lar), ermA was part of the transposon Tn554, ²⁵ integrated into their chromosomes. The insertion of Tn554 at different sites was observed. However, in the ST398 S. aureus isolate Sau-3221Lar, the ermA gene was carried by the transposon Tn6133. Tn6133 was composed of Tn554 with an integrated 4,787-bp DNA sequence encoding VgaE conferring streptogramin A, pleuromutilin, and lincosamide resistance. ²⁶ Tn6133 has been previously detected in several ST398 MRSA isolates. ²⁶ Screening by PCR and sequencing identified that all ermA-positive S. aureus, except isolate Sau-3221Lar, harbored the ermA-carrying Tn554 transposon.

Furthermore, WGS data showed that both ermC-positive S. aureus isolates (ST1 S. aureus Sau-2716Lar and ST121 S. aureus Sau-3893Lar), sequenced during this study, included small plasmids (pSau-2716Lar and pSau-3893Lar) of ∼3 kb (Fig. 1). Both small plasmids comprised the repL and ermC genes, and the insertion sequence IS257. Previous studies have reported the presence of small (2.3–2.5 kb) ermC-carrying plasmids, like pNE131 ²⁷ in staphylococcal isolates. ²⁸ Screening by PCR and sequencing confirmed that all ermC-positive S. aureus harbored repL-associated plasmids, which carried the ermC gene.

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FIG. 1.

Linear maps of mobile elements carrying erm (A) ermA, (B) ermC. genes. Arrows show the direction of transcription of ORFs. ORFs are classified by function into different groups. Homologous segments (representing ≥99% sequence identity) are indicated by light gray shading, whereas gray shading with dots shows inverted homologous segments. The features shown are not to scale. ORFs, open reading frames.

Further analysis of WGS data

Analysis of WGS data by ResFinder 3.0 tool (https://cge.cbs.dtu.dk/services/ResFinder/) ²⁹ revealed that the majority of the sequenced isolates included additional genes for resistance to aminoglycosides, β-lactams, fluoroquinolones, fusidic acid, and tetracyclines (Table 4). The presence of mecA, blaZ, aph(3′)-III, aadD, aac(6′)-aph(2″), dfrG, tetK, tetM, fusB, and cat genes was confirmed by the antimicrobial resistant phenotypes of the isolates harboring those genes; only the presence of norA and fosD genes was not associated with the phenotypes. However, susceptibility to spectinomycin was not tested to clarify the expression of ant(9)-Ia gene in the respective isolates. S. aureus isolates that were assigned to ST225 harbored the highest number of resistance genes, explaining the MDR phenotypes of isolates belonging to this clone.

Additionally, analysis of WGS data by VirulenceFinder 1.5 tool ³⁰ showed that all sequenced were positive for the presence of several virulence genes (Table 4), which could be implicated in colonization and later infection of the host. ³¹ Diverse profiles of virulence factors were observed in isolates belonging to different clones. ST225 S. aureus isolates that were the most prevalent in our setting carried genes encoding Gamma-hemolysin (HlgA, HlgB, HlgC), Hlb hemolysin, which permeabilize cell membranes and lyse macrophages and lymphocytes and alter platelet morphology ³¹ and leukocidins (LukD and LukE) forming pores in leukocyte membranes that results in cell death. ³² Also, ST225 isolates encoded the staphylokinase, enterotoxins (SED, SER, SEG, SEJ, SEI, SEM, SEN, SEO, SEU), exhibiting superantigen activity that deregulates the immune system of the host, the staphylococcal inhibitor of complement, and metalloprotease aureolysin (Aur). Previous studies have shown that different combinations of virulence factors are carried by different phages. ³³