Linezolid-resistant Enterococcus faecium strains isolated from one hospital in Poland -commensals or hospital-adapted pathogens?

Abstract

One of the most pressing problems of enterococci infections is occurring resistance to linezolid, which is an antibiotic used in the treatment of infections caused by vancomycin-resistant strains (VRE). The main objective of our research was to investigate the relationship of 19 linezolid-resistant E. faecium isolates from 18 patients hospitalized at Clinical Hospital in Gdansk (Poland). One of the LZDREF was isolated in 2003 (K2003), and another 18 were collected from 2013 to 2017. Genotyping with PCR MP method indicated 14 main unrelated genetic profiles and no association with K2003 strain. Two isolates with the same genotype and genetically closely related two sub-types (2 isolates for each sub-type) were hospital-derived colonizations of patients. The other unrelated genotypes were discussed in the context of colonization, nosocomial infections, and commensal origin, taking into account prior exposure to linezolid. We determined the presence of a point mutation G2576T in six loci of 23S rDNA. There was also a significant correlation (p<0.0015) between the presence of MIC>32 value and the presence of G2576T point mutation on the sixth rrn. We also detected 5 virulence genes for all isolates: gelE, cylA, asa1, hyl, esp. Correlation (p≤0.0001) was observed between the presence of gelE gene encoding gelatinase and two other genes: cylA and asa1 encoding cytolysin and collagen binding protein responsible for aggregation of bacterial cells, respectively. Significant correlation was also observed between asa1 and cfr genes encoding 23S rRNA rybonuclease responsible for resistance to PhLOPSA antibiotics (p = 0.0004). The multidimensional analysis has also shown the correlation between cfr gene and GI-tract (p = 0, 0491), which suggests horizontal gene transfer inside the gut microbiota and the risk of colonization with linezolid-resistant strains without previously being treated with the antibiotic. The patient could have been colonized with LZDRVREF strains which in the absence of competitive microbiota quickly settle in ecological niches favourable for them and pose a risk for the patient.

Fig 1. The phylogenetic relationships for clinical LZD^REF isolates, characteristics of linezolid resistance and virulence profiles.

The scale value represents 0.06 parameter substitution per site. Legend: Is—isolate; Sp—specimen;RS—rectum swab; SBS—bedsore swab; G-Type–genotypes designation as (A-N); CopyMut—copy number with a mutation; cfr—gene coding for 23S rRNA methyltransferase; Vf—virulence factors: esp—enterococcal surface protein; asa1—aggregation substance; cylA—cytolysin; gelE—gelatinase; hyl—hyalorunidase; T-LZD—treatment previously with linezolid.

Fig 2. Real-time PCR and high-resolution melt analysis for detection of point mutations responsible for linezolid-susceptible.

(A) High-resolution melting profiles for 23S rDNA fragment gene/s with or not G2576T mutation/s. Legend: grey colour–wild strains of E. faecium; black colour–strains with G2576T mutation/s. (B) A graph generated base on HRM—real time PCR, showing the assignment of the strains to clusters, each cluster is surrounded by an ellipse corresponding to a different genotype, with (bigger cluster) or not (smaller cluster) G2576T mutation.

Fig 3. Significant positive and negative correlations between 14 measured parameters describing linezolid-resistant clinical Enterococcus faecium isolates.

Legend: Values highlighted on grey background are statistically significant (p<0.05) and positively correlated. Values distinguished on black background are negatively correlated. Values are calculated using linear correlation statistic and uncorrected p-values (Past software v. 3.25). GI- tract–gastro-intestinal tract; pLZD–patients previously treated with linezolid; AML–patients with acute myeloid leukemia; cfr–gene coding for 23S rRNA methyltransferase; LZD–linezolid; G2576U_1 e.t.c–mutation in copy number 1 e.t.c; 1C_LZD—mutated 1 copy e.t.c; genes of virulence factors: esp—gene encoding enterococcal surface protein; asa1—gene encoding aggregation substance; cylA- gene encoding cytolysin; gelE- gene encoding gelatinase; hyl- gene encoding hyalorunidase.

Fig 4. Pseudo-phylogenetic tree presenting similarity between the tested parameters.

The correlation was examined between: virulence genes, resistance to antibiotics, location of G2576U mutation in six rrn operons, frequency of acute myeloid leukemia disease among patients, and gender of the patients. The tree was constructed by using the neighbor-joining method from 0–1 matrix. Description of parameters analyzed like in Fig 3.