Emergence of optrA-mediated linezolid resistance in clinical isolates of Enterococcus faecalis from Argentina.

OBJECTIVES: The aim of this study was to characterize the first 14 optrA-carrying linezolid resistant E. faecalis clinical isolates recovered in seven Argentinian hospitals between 2016 and 2021. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. METHODS: The isolates were phenotypically and genotypically characterized. Susceptibility to 13 antimicrobial agents was performed; clonal relationship was assessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Data provided by the whole-genome sequencing were used for identification of sequence types, antimicrobial resistance genes, optrA variants, phylogenetic tree, and mobile genetic elements responsible to the dissemination of these strains. RESULTS: All the optrA-carrying E. faecalis isolates were multidrug-resistant and harboured several antimicrobial resistance genes. They carried three optrA variants and belonged to different lineages; however, three of them belonged to the hyperepidemic CC16. Mobile genetic elements were detected in all the isolates. The analysis of the optrA flanking region suggests the plasmidic localization in most of the isolates. CONCLUSIONS: To the best of our knowledge, this is the first report of optrA-mediated linezolid resistance in Argentina. The emergence and dissemination of the optrA genes in clinical E. faecalis isolates are of concern and highlights the importance of initiating the antimicrobial surveillance of Enterococcus spp. under a One Health strategy.

Isolation and first draft genome sequence of a linezolid-dependent Staphylococcus aureus clinical strain.

Background: Antibiotic-dependent pathogenic bacteria are sporadically isolated from patients that received prolonged antibiotic treatments. Evolution of antibiotics dependence and its clinical implications are scarcely studied. Materials & methods: A linezolid-dependent Staphylococcus aureus strain was isolated from a cystic fibrosis patient. A draft genome sequence was obtained and searched for known antibiotics resistance determinants and virulence factors. Results: The genome was assembled into 79 contigs for a total of 2.83 Mbp. This strain is a sequence type 5 methicillin-resistant Staphylococcus aureus with a type I SCCmec cassette also conserving the Panton-Valentine leukocidin. The G2576T substitution, conferring linezolid resistance, was harbored by all five copies of the 23S rRNA. Conclusion: The linezolid-dependent strain is related to a strain circulating in Latin America that acquired a mutation conferring linezolid resistance.

Molecular epidemiology of coagulase-negative bloodstream isolates: detection of Staphylococcus epidermidis ST2, ST7 and linezolid-resistant ST23

Abstract The mechanisms contributing to persistence of coagulase-negative staphylococci are diverse; to better understanding of their dynamics, the characterization of nosocomial isolates is needed. Our aim was to characterize phenotypic and molecular characteristics of Staphylococcus epidermidis and Staphylococcus haemolyticus human blood isolates from two tertiary care hospitals in Mexico, the Hospital Universitario in Monterrey and the Hospital Civil in Guadalajara. Antimicrobial susceptibility was determined. Biofilm formation was assessed by crystal violet staining. Detection of the ica operon and Staphylococcal Cassette Chromosome mec typing were performed by PCR. Clonal relatedness was determined by Pulsed-fiel gel electrophoresis and Multi locus sequence typing. Methicillin-resistance was 85.5% and 93.2% for S. epidermidis and S. haemolyticus, respectively. Both species showed resistance >70% to norfloxacin, clindamycin, levofloxacin, trimethoprim/sulfamethoxazole, and erythromycin. Three S. epidermidis and two S. haemolyticus isolates were linezolid-resistant (one isolate of each species was cfr+). Most isolates of both species were strong biofilm producers (92.8% of S. epidermidis and 72.9% of S. haemolyticus). The ica operon was amplified in 36 (43.4%) S. epidermidis isolates. SCCmec type IV was found in 47.2% of the S. epidermidis isolates and SCCmec type V in 14.5% of S. haemolyticus isolates. No clonal relatedness was found in either species. Resistance to clindamycin, levofloxacin, erythromycin, oxacillin, and cefoxitin was associated with biofilm production for both species (p < 0.05). A G2576T mutation in 23S rRNA gene was detected in an S. haemolyticus linezolid-resistant isolate. All linezolid-resistant S. epidermidis isolates belonged to ST23; isolate with SCCmec type IV belonged to ST7, and isolate with SCCmec type III belonged to ST2. This is the first report of ST7 in Mexico. There was a high genetic diversity in both species, though both species shared characteristics that may contibute to virulence.

Virulence and resistance pattern of a novel sequence type of linezolid-resistant Enterococcus faecium identified by whole-genome sequencing.

Empirical use of linezolid has been advocated in neutropenic febrile patients colonised by vancomycin-resistant enterococci (VRE) because of the risk of bloodstream infection (BSI). This study aimed to genetically describe a vancomycin-resistant Enterococcus faecium (VREfm) BSI isolate resistant to linezolid (VRLRE) in a patient previously colonised by VREfm and to determine the incidence of colonisation and infection by VREfm in a bone marrow transplant unit over a 10-year period. Data for VREfm colonisation and infection were evaluated. PCR for the vanA and vanB genes, pulsed-field gel electrophoresis (PFGE) and microdilution antimicrobial susceptibility testing (vancomycin, teicoplanin, linezolid and aminoglycosides) were performed. Three isolates, including the VRLRE, were selected for whole-genome sequencing by Ion Torrent™, with E. faecium CP006620-Aus0085 used as a reference. Eighty-seven VREfm were analysed; all were linezolid-susceptible and harboured vanA, except for one blood isolate from a febrile neutropenic patient colonised by VREfm who received linezolid for 12 days and developed a BSI by VRLRE (linezolid MIC≥8µg/mL). Linezolid resistance was associated with a G2576T mutation in the 23SrRNA gene. PFGE analysis demonstrated that the 87 isolates belonged to four major clusters; however, the VRLRE presented only 50% similarity. Three sequence types (STs) were identified: ST412 (the predominant clone, which was more virulent compared with the other isolates); ST478 (linezolid-susceptible VREfm); and a novel ST named ST987 (VRLRE). SNP analysis showed a higher similarity between linezolid-susceptible VREfm and the predominant clone compared with VRLRE. VRLRE presented a G2576T mutation and belonged to a novel ST (ST987).

Molecular epidemiology of coagulase-negative bloodstream isolates: detection of Staphylococcus epidermidis ST2, ST7 and linezolid-resistant ST23.

The mechanisms contributing to persistence of coagulase-negative staphylococci are diverse; to better understanding of their dynamics, the characterization of nosocomial isolates is needed. Our aim was to characterize phenotypic and molecular characteristics of Staphylococcus epidermidis and Staphylococcus haemolyticus human blood isolates from two tertiary care hospitals in Mexico, the Hospital Universitario in Monterrey and the Hospital Civil in Guadalajara. Antimicrobial susceptibility was determined. Biofilm formation was assessed by crystal violet staining. Detection of the ica operon and Staphylococcal Cassette Chromosome mec typing were performed by PCR. Clonal relatedness was determined by Pulsed-fiel gel electrophoresis and Multi locus sequence typing. Methicillin-resistance was 85.5% and 93.2% for S. epidermidis and S. haemolyticus, respectively. Both species showed resistance >70% to norfloxacin, clindamycin, levofloxacin, trimethoprim/sulfamethoxazole, and erythromycin. Three S. epidermidis and two S. haemolyticus isolates were linezolid-resistant (one isolate of each species was cfr+). Most isolates of both species were strong biofilm producers (92.8% of S. epidermidis and 72.9% of S. haemolyticus). The ica operon was amplified in 36 (43.4%) S. epidermidis isolates. SCCmec type IV was found in 47.2% of the S. epidermidis isolates and SCCmec type V in 14.5% of S. haemolyticus isolates. No clonal relatedness was found in either species. Resistance to clindamycin, levofloxacin, erythromycin, oxacillin, and cefoxitin was associated with biofilm production for both species (p<0.05). A G2576T mutation in 23S rRNA gene was detected in an S. haemolyticus linezolid-resistant isolate. All linezolid-resistant S. epidermidis isolates belonged to ST23; isolate with SCCmec type IV belonged to ST7, and isolate with SCCmec type III belonged to ST2. This is the first report of ST7 in Mexico. There was a high genetic diversity in both species, though both species shared characteristics that may contibute to virulence.

Linezolid-resistant Staphylococcus haemolyticus and Staphylococcus hominis: single and double mutations at the domain V of 23S rRNA among isolates from a Rio de Janeiro hospital.

In this work, the molecular and phenotypic antimicrobial resistance and clonal diversity of 10 linezolid-resistant Staphylococcus spp. isolates were investigated. The 7 Staphylococcus haemolyticus isolates presented Staphylococcal cassete chromosome mec (SCCmec) V and belonged to the same pulsed-field gel electrophoresis pulsotype. Their MICs for oxacillin, vancomycin, and linezolid were ≥ 256 µg/mL, 1-4 µg/mL, and 8-16 µg/mL, respectively. The 3 S. hominis presented MIC values 32 to >256 µg/mL, 2-4 µg/mL, and 12-24 µg/mL, and all carried the nontypeable SCCmec (ccr1 + mecA class) and belonged to 2 different genotypes. The cfr gene was not found, but the mutation G2603T was detected in S. haemolyticus and C2190T and G2603T in Staphylococcus hominis in 23S rRNA. This study demonstrates the spread of a linezolid-resistant S. haemolyticus genotype and, for the first time, describes the mutation C2190T among S. hominis isolates with a double mutation in Brazil.