Mechanisms Of Linezolid Resistance Research Articles

Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China

BackgroundRecently, linezolid-resistant staphylococci have become an emerging problem worldwide. Understanding the mechanisms of resistance, molecular epidemiology and transmission of linezolid-resistant CoNS in hospitals is very important.MethodsThe antimicrobial susceptibilities of all isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were determined using whole-genome sequencing and PCR.ResultsAll the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had prior linezolid exposure. Most S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated most linezolid-resistant CoNS strains were genetically related. In this study, we revealed that distinct CoNS strains have different mechanisms of linezolid resistance. Among ST22-type S. epidermidis, acquisition of the T2504A and C2534T mutations in the V domain of the 23 S rRNA gene, as well as mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S), were linked to the development of linezolid resistance. In S. cohnii isolates, cfr, S158Y and D159Y mutations in the ribosomal protein L3 were detected. Additionally, emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, G2576T and C2104T mutations, M156T change in L3 protein, and I188S change in L4 protein were found in S. capitis isolates.ConclusionThe emergence of linezolid-resistant CoNS in the environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.

Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China

BackgroundInfections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022.MethodsEnterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis.ResultsThe optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains.ConclusionsOur study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Persistence and evolution of linezolid- and methicillin-resistant Staphylococcus epidermidis ST2 and ST5 clones in an Italian hospital

Staphylococcus epidermidis is a member of the human skin microbiome. However, in recent decades, multidrug-resistant and hospital-adapted S. epidermidis clones are increasingly involved in severe human infections associated with medical devices and in immunocompromised patients. In 2016, we reported that a linezolid- and methicillin-resistant S. epidermidis ST2 clone, bearing the G2576T mutation, was endemic in an Italian hospital since 2004. This study aimed to retrospectively analyse 34 linezolid- and methicillin-resistant S. epidermidis (LR-MRSE) strains collected from 2018 to 2021 from the same hospital. LR-MRSE were typed by Pulsed-Field Gel Electrophoresis and multilocus sequence typing and screened for transferable linezolid resistance genes. Representative LR-MRSE were subjected to whole-genome sequencing (WGS) and their resistomes, including the presence of ribosomal mechanisms of linezolid resistance and of rpoB gene mutations conferring rifampin resistance, were investigated. ST2 lineage was still prevalent (19/34; 55.9%), but, over time, ST5 clone has been widespread too (15/34; 44.1%). Thirteen of the 34 isolates (38.2%) were positive for the cfr gene. Whole-genome sequencing analysis of relevant LR-MRSE displayed complex resistomes for the presence of several acquired antibiotic resistance genes, including the SCCmec type III (3A) and SCCmec type IV (2B) in ST2 and ST5 isolates, respectively. Bioinformatics and polymerase chain reaction (PCR) mapping also showed a plasmid-location of the cfr gene and the occurrence of previously undetected mutations in L3 (ST2 lineage) and L4 (ST3 lineage) ribosomal proteins and substitutions in the rpoB gene. The occurrence of LR-MRSE should be carefully monitored in order to prevent the spread of this difficult-to-treat pathogen and to preserve the efficacy of linezolid.

Multiple mechanisms of linezolid resistance in Staphylococcus haemolyticus detected by whole-genome sequencing.

Introduction. Linezolid is an effective therapeutic option for treating severe infections caused by multidrug-resistant Gram-positive organisms. Several mechanisms have been reported to be responsible for resistance to this antibiotic.Hypothesis or Gap Statement. Although several mechanisms of linezolid resistance have been reported in Staphylococcus haemolyticus, the prevalence and potential for horizontal transfer of resistance genes have not been fully characterized, particularly among S. haemolyticus isolates from India.Aim. To perform whole-genome sequencing (WGS) of linezolid-resistant S. haemolyticus isolates to characterize the resistance mechanisms.Methodology. WGS was performed for 16 linezolid-resistant S. haemolyticus isolates to check for the presence of cfr, optrA and poxtA genes and mutations in 23S rRNA and ribosomal proteins (L3, L4 and L22) that are possible mechanisms implicated in linezolid resistance. Sequence types were identified using MLST finder. The minimum inhibitory concentration (MIC) of linezolid was determined using the E-test method. Polymerase chain reaction (PCR) was carried out for the detection of the cfr gene.Results. The study documented three different mechanisms of linezolid resistance in S. haemolyticus. Thirteen of the 16 isolates were phenotypically resistant to linezolid, of which 12 were positive for the cfr gene. The G2603T mutation in 23S rRNA was found in the majority of the isolates (n=13). Ten isolates had the R138V mutation in L3 ribosomal protein. Twelve isolates with the cfr gene in combination with either G2603T or R138V mutations displayed extremely high MIC values. Surprisingly, three phenotypically sensitive isolates were found to be positive for the cfr gene but negative for other resistance mechanisms. Importantly, in almost half of the isolates the cfr gene was present on a plasmid. ST3 and ST1 were found to be the predominant sequence types.Conclusion. All phenotypically resistant isolates exhibited two or three linezolid resistance mechanisms. The cfr gene was found on plasmids in many isolates, demonstrating its potential for horizontal transfer to more pathogenic organisms.

Mechanisms of Linezolid Resistance in Mycobacteria.

Mycobacteria form some of the most notorious and difficult-to-treat bacterial pathogens. As a group, they are intrinsically resistant to many commonly used antibiotics, such as tetracyclines and beta-lactams. In addition to intrinsic resistances, acquired multidrug resistance has also been observed and documented in Mycobacterium tuberculosis (MTB), Mycobacterium leprae and non-tuberculous mycobacteria (NTM). To combat multidrug resistant infections by these pathogens, innovative antimicrobials and treatment regimens are required. In this regard, linezolid, an oxazolidinone introduced for clinical use just two decades ago, was added to the therapeutic armamentarium for drug-resistant mycobacteria. It exhibits antibacterial activity by binding to the 50S ribosomal subunit and inhibiting protein synthesis. Unfortunately, linezolid resistance has now been documented in MTB and NTM, in many parts of the world. Most linezolid-resistant mycobacterial strains show mutations in the ribosome or related genes, such as in the rplC, rrl and tsnR genes. Non-ribosomal mechanisms appear to be rare. One such mechanism was associated with a mutation in fadD32, which encodes a protein that plays an important role in mycolic acid synthesis. Mycobacterial efflux proteins have also been implicated in linezolid resistance. This review summarises current knowledge of genetic determinants of linezolid resistance in mycobacteria, with the aim of contributing information that could facilitate the discovery of new therapeutic approaches to overcome, delay or avoid further developments of drug resistance among these important pathogens.

Mutations in Genes Encoding 23S rRNA and FadD32 May be Associated with Linezolid Resistance in Mycobacteroides abscessus.

Linezolid is one of the antibiotics used to treat the Mycobacteroides abscessus infection. However, linezolid-resistance mechanisms of this organism are not well understood. The objective of this study was to identify possible linezolid-resistance determinants in M. abscessus through characterization of step-wise mutants selected from a linezolid-susceptible strain, M61 (minimum inhibitory concentration [MIC]: 0.25 mg/L). Whole-genome sequencing and subsequent PCR verification of the resistant second-step mutant, A2a(1) (MIC: >256 mg/L), revealed three mutations in its genome, two of which were found in the 23S rDNA (g2244t and g2788t) and another one was found in a gene encoding the fatty-acid-CoA ligase FadD32 (c880t→H294Y). The 23S rRNA is the molecular target of linezolid and mutations in this gene are likely to contribute to resistance. Furthermore, PCR analysis revealed that the c880t mutation in the fadD32 gene first appeared in the first-step mutant, A2 (MIC: 1 mg/L). Complementation of the wild-type M61 with the pMV261 plasmid carrying the mutant fadD32 gene caused the previously sensitive M61 to develop a reduced susceptibility to linezolid (MIC: 1 mg/L). The findings of this study uncovered hitherto undescribed mechanisms of linezolid resistance in M. abscessus that may be useful for the development of novel anti-infective agents against this multidrug-resistant pathogen.

Linezolid resistance: detection of the cfr(B) gene in French clinical MRSA strains.

To describe two linezolid-resistant MRSA strains carrying the cfr(B) gene detected in the French National Reference Centre for staphylococci. Two linezolid-resistant MRSA strains isolated from cystic fibrosis patients in two different French hospitals in 2017 and 2019 were examined to explore the mechanisms of linezolid resistance. Antimicrobial susceptibility was tested using broth microdilution and gradient strips. The genetic determinants of linezolid resistance were assessed by a multiplex PCR targeting cfr/cfr(B), optrA and poxtA genes, by amplification and sequencing of individual 23S rRNA genes and by WGS using both Illumina and Nanopore technologies. The two MRSA strains were resistant to linezolid but susceptible to tedizolid, and PCR-positive for cfr/cfr(B). The WGS analysis indicated that they belonged to two different STs (ST8-MRSA-IV and ST5382-MRSA-IV) and that they both harboured the cfr(B) gene on the same 9.7 kb Tn6218-like chromosomal transposon, a finding only previously reported in Enterococcus sp. and Clostridioides difficile. To the best of our knowledge, this is the first description of the presence of cfr(B) in staphylococci, more specifically in linezolid-resistant MRSA strains. This finding illustrates the risk of horizontal intergenus transfer of oxazolidinone resistance genes in Staphylococcus aureus and highlights the need to monitor such emergence in this species.

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer.

Emergence and Genomic Characterization of the First Reported optrA-Carrying Linezolid-Resistant Enterococci Isolated from Retail Broiler Meat in the United Arab Emirates

The foodborne transfer of resistant genes from enterococci to humans and their tolerance to several commonly used antimicrobials are of growing concern worldwide. Linezolid is a last-line drug for managing complicated illnesses resulting from multidrug-resistant Gram-positive bacteria. The optrA gene has been reported in enterococci as one of the acquired linezolid resistance mechanisms. The present study uses whole-genome sequencing analysis to characterize the first reported isolates of linezolid-resistant E. faecium (n = 6) and E. faecalis (n = 10) harboring the optrA gene isolated from samples of supermarket broiler meat (n = 165) in the United Arab Emirates (UAE). The sequenced genomes were used to appraise the study isolates’ genetic relatedness, antimicrobial resistance determinants, and virulence traits. All 16 isolates carrying the optrA gene demonstrated multidrug-resistance profiles. Genome-based relatedness classified the isolates into five clusters that were independent of the isolate sources. The most frequently known genotype among the isolates was the sequence type ST476 among E. faecalis (50% (5/10)). The study isolates revealed five novel sequence types. Antimicrobial resistance genes (ranging from 5 to 13) were found among all isolates that conferred resistance against 6 to 11 different classes of antimicrobials. Sixteen different virulence genes were found distributed across the optrA-carrying E. faecalis isolates. The virulence genes in E. faecalis included genes encoding invasion, cell adhesion, sex pheromones, aggregation, toxins production, the formation of biofilms, immunity, antiphagocytic activity, proteases, and the production of cytolysin. This study presented the first description and in-depth genomic characterization of the optrA-gene-carrying linezolid-resistant enterococci from retail broiler meat in the UAE and the Middle East. Our results call for further monitoring of the emergence of linezolid resistance at the retail and farm levels. These findings elaborate on the importance of adopting a One Health surveillance approach involving enterococci as a prospective bacterial indicator for antimicrobial resistance spread at the human–food interface.

Mechanisms of linezolid resistance in Staphylococcus capitis with the novel mutation C2128T in the 23S rRNA gene in China

PurposeThe objective of this study was to investigate the molecular characteristics and potential resistance mechanisms of linezolid-resistant (LZR) Staphylococcus capitis isolates from a tertiary hospital in China.MethodsS. capitis isolates were obtained from clinical patient specimens; three of the isolates came from blood cultures and one from the hydrothorax. The agar dilution and E-test methods were used to identify antibiotic resistance. The chloramphenicol-florfenicol resistance (cfr) gene carrier status of the strains was determined by PCR. Whole-genome sequencing (WGS) was used to identify point mutations and L3, L4, and L22 mutations and to study the genetic environment of the cfr gene and the relationships between strains.ResultsThe 4 isolates obtained in this study were all linezolid-resistant Staphylococcus strains. A similar of susceptibility profile pattern was observed in all four S. capitis strains, each of which exhibited a multidrug-resistant phenotype. A potentially novel mutation, C2128T, was identified, and the cfr genes of S. capitis strains were all positive. Additionally, the same mutations (C2128T and G2600T) were identified in all 23S rRNA sequences of the isolates, whereas mutations were lacking in the L3, L4, and L22 ribosomal proteins. The genetic environments surrounding cfr were identical in all four isolates. A schematic diagram of the phylogenetic tree showed that they were closely related to AYP1020, CR01, and TW2795, and a total of seven drug resistance genes were identified in these strains.ConclusionsThe study indicated that the resistance of the Staphylococcus capitis strains to linezolid was caused by multiple mechanisms, and a potential novel mutation, C2128T, that may have an impact on bacterial resistance was identified.

Linezolid-Resistant Enterococcus spp. Isolates from Foods of Animal Origin-The Genetic Basis of Acquired Resistance.

Enterococci are important opportunistic pathogens with the capacity to acquire and spread antibiotic resistance. At present, linezolid-resistant enterococci (LRE) pose a great challenge. Linezolid is considered as a last resort antibiotic in the treatment of enterococcal infections, so it is important to monitor the occurrence of LRE in various environments. The aim of this study was to define the genetic mechanisms of linezolid resistance in enterococci (E. faecalis, E. faecium, E. hirae, E. casseliflavus) isolated from foods of animal origin (n = 104). Linezolid resistance (LR) was shown by 26.9% of isolates. All of them displayed linezolid MICs of 8–32 µg/mL, and 96.4% of them were multidrug multidrug-resistant. The most common acquired linezolid resistance gene in LR isolates was poxtA (64%), followed by optrA (28%) and cfr (12%). According to the authors’ knowledge, this research is the first to indicate the presence of the cfr gene among isolates from food. In 28.6% of the isolates, the point mutation G2576T in the V domain of the 23S rRNA was responsible for linezolid resistance. All isolates harbored the wild-type rplC, rplD and rplV genes. The obtained results indicate that linezolid resistance among enterococci in animal-derived food may result from various genetic mechanisms. The most worrying is that this resistance is encoded on mobile genetic elements, so there is a risk of its rapid transmission, even despite the lack of selective pressure resulting from the use of antibiotics.

Sentinel Surveillance Reveals Emerging Daptomycin-Resistant ST736 Enterococcus faecium and Multiple Mechanisms of Linezolid Resistance in Enterococci in the United States.

Enterococcus faecalis and faecium with resistance to daptomycin and/or linezolid are emerging globally. We present the genomic characterization of daptomycin- and linezolid-resistant E. faecalis and E. faecium surveillance isolates from the United States, 2013–2016. Daptomycin resistance was low among E. faecalis (2/364, 0.5%) and E. faecium (17/344, 5%). The majority (71%, 12/17) of daptomycin-resistant E. faecium isolates belonged to the emerging ST736 clone and contained mutations in liaFSR and cls previously associated with resistance. However, 1/2 E. faecalis and 3/17 E. faecium did not contain these mutations previously associated with daptomycin resistance. Linezolid resistance was rare among E. faecalis (1/364, 0.3%) and E. faecium (2/344, 0.6%). These two E. faecium isolates, one of which was also resistant to daptomycin and vancomycin, contained the 23S rRNA nucleotide mutation (G2576T) associated with linezolid resistance. Long-read sequencing revealed the linezolid-resistant E. faecalis isolate contained chromosomal- and plasmid-encoded copies of optrA. The chromosomal optrA was located on the recently described Tn6674 multiresistance transposon. The second copy of optrA was encoded on an ∼65 kb mosaic plasmid, with component regions sharing high sequence identity to optrA-encoding multiresistance plasmids of animal origin. The optrA-encoding plasmid contained open reading frames predicted to encode proteins associated with a pheromone-responsive plasmid transfer system, and filter mating experiments confirmed the plasmid was conjugative. Continued surveillance of enterococci is necessary to assess the prevalence and trends of daptomycin and linezolid resistance in the United States, characterize resistance mechanisms and how they transfer, and monitor for emerging sequence types associated with resistance.

Investigation of radezolid interaction with non-canonical chloramphenicol binding site by molecular dynamics simulations

Radezolid is a promising antibiotic of oxazolidinone family, which is able to overcome effect of some linezolid resistance mechanisms of bacterial ribosomes. The structure of the radezolid complex with ribosomes was never published but, by analogy with linezolid, it is considered to prevent the binding of aminoacyl–tRNA to the A–site of the ribosome large subunit. However, as with linezolid, it can be assumed that radezolid binds to the alternative binding site existing in the A,A/P,P–ribosome. In the present article we have investigated this issue by molecular dynamics simulations and proposed the structure of the radezolid complex with a E. coli ribosome, which is consistent with available data of biochemical investigations of radezolid action.

Linezolid-resistant Enterococcus faecalis ST16 harbouring optrA on a Tn6674-like element isolated from surface water

The objective of this work was to characterise an optrA-carrying Enterococcus faecalis ST16 isolate recovered from a river water sample in Switzerland. Linezolid-resistant E. faecalis F102 was recovered from surface water in Switzerland and was subjected to comprehensive genotypic characterisation and analysis of the genetic environment of the oxazolidinone/phenicol resistance gene optrA. Whole-genome sequencing (WGS) was performed to detect linezolid resistance mechanisms, including mutations in 23S rRNA and ribosomal protein genes as well as acquired resistance genes. The isolate was further characterised by multilocus sequence typing (MLST) and identification of virulence genes. WGS detected the presence of optrA identical to the original optrA gene from E. faecalis E349. Analysis of the genetic environment revealed the association of optrA with fexA and a Tn6674-like transposon in co-existence with spc and erm(A) resistance genes. Sequence alignment indicated that the genetic environment of optrA was identical to a Tn6674-like variant from E. faecalis previously isolated from diseased and healthy humans and food-producing animals in the Asia-Pacific region. Enterococcus faecalis F102 did not contain any mutations in 23S rRNA or in genes encoding ribosomal proteins L3, L4 and L22. A total of 14 other resistance genes and 16 virulence genes were detected. Enterococcus faecalis F102 was assigned in silico to ST16. The spread of optrA-carrying E. faecalis ST16 with a high pathogenic potential in surface water is a worrisome aspect from a public-health perspective.

Detection of a cfr-positive MRSA CC398 strain in a pig farmer in Spain

ObjectiveTo characterize one linezolid- and methicillin-resistant Staphylococcus aureus (MRSA) isolate recovered from a nasal sample of a pig farmer patient. MethodsThe detection of linezolid resistance mechanisms was performed by PCR and sequencing. The antimicrobial resistance and virulence profile was investigated, and the molecular typing was performed by molecular techniques. The transference of cfr gene was assessed by conjugation experiments and its genetic environment was investigated by specific PCRs. ResultsThe linezolid-resistant MRSA isolate was typed as t011-ST398/CC398-SCCmecV-agrI and carried the cfr gene. The isolate was multidrug-resistant but lacked the virulence genes studied. The cfr gene was co-located with the fexA gene on a Tn558 variant and was successfully transferred by conjugation. ConclusionWe report the first description of LA-MRSA-CC398 carrying the cfr gene in Spain. This finding highlights the importance of surveillance programmes to determine the presence and spread of the cfr gene in the livestock and clinical settings.

Spread of Linezolid-Resistant Enterococcus spp. in Human Clinical Isolates in the Czech Republic.

The aim of this study was to map and investigate linezolid resistance mechanisms in linezolid-resistant enterococci in the Czech Republic from 2009 to 2019. Altogether, 1442 isolates of Enterococcus faecium and Enterococcus faecalis were examined in the National Reference Laboratory for Antibiotics. Among them, 8% of isolates (n = 115) were resistant to linezolid (E. faecium/n = 106, E. faecalis/n = 9). Only three strains of E. faecium were resistant to tigecycline, 72.6% of isolates were resistant to vancomycin. One isolate of E. faecium harbored the cfr gene. The majority (87%, n = 11) of E. faecium strains were resistant to linezolid because of the mutation G2576T in the domain V of the 23S rRNA. This mutation was detected also in two strains of E. faecalis. The presence of the optrA gene was the dominant mechanism of linezolid resistance in E. faecalis isolates. None of enterococci contained cfrB, poxtA genes, or any amino acid mutation in genes encoding ribosomal proteins. No mechanism of resistance was identified in 4 out of 106 E. faecium linezolid resistant isolates in this study. Seventeen sequence types (STs) including four novel STs were identified in this work. Clonal complex CC17 was found in all E. faecium isolates.

Emergence of cfr-Mediated Linezolid Resistance in Staphylococcus aureus Isolated from Pig Carcasses.

Altogether, 2547 Staphylococcus aureus isolated from cattle (n = 382), pig (n = 1077), and chicken carcasses (n = 1088) during 2010–2017 were investigated for linezolid resistance and were further characterized using molecular methods. We identified linezolid resistance in only 2.3% of pig carcass isolates. The linezolid-resistant (LR) isolates presented resistance to multiple antimicrobials, including chloramphenicol, clindamycin, and tiamulin. Molecular investigation exhibited no mutations in the 23S ribosomal RNA. Nevertheless, we found mutations in ribosomal proteins rplC (G121A) and rplD (C353T) in one and seven LR strains, respectively. All the LR isolates carried the multi-resistance gene cfr, and six of them co-carried the mecA gene. Additionally, all the LR isolates co-carried the phenicol exporter gene, fexA, and presented a high level of chloramphenicol resistance. LR S. aureus isolates represented 10 genotypes, including major genotypes ST433-t318, ST541-t034, ST5-t002, and ST9-t337. Staphylococcal enterotoxin and leukotoxin-encoding genes, alone or in combination, were detected in 68% of LR isolates. Isolates from different farms presented identical or different pulsed-field gel electrophoresis patterns. Collectively, toxigenic and LR S. aureus strains pose a crisis for public health. This study is the first to describe the mechanism of linezolid resistance in S. aureus isolated from food animal products in Korea.

Prevalence and mechanisms of linezolid resistance among staphylococcal clinical isolates from Egypt

The emergence of methicillin-resistant staphylococci necessitated the search for alternative agents as linezolid, introduced to treat infections due to multidrug-resistant bacteria. Linezolid resistance has since emerged, yet its global prevalence remains low. In Egypt, little is known about the situation. We investigated the prevalence and mechanisms of resistance among Egyptian staphylococcal clinical isolates. Linezolid resistance among 232 staphylococcal isolates obtained from Alexandria Main Hospitals between 2011 and 2016 was assessed using disc diffusion and minimum inhibitory concentration. Resistant isolates were checked for cfr presence using polymerase chain reaction. The V domain of different alleles of 23S rRNA gene was investigated for mutations. Selection for linezolid-resistant mutants was performed in vitro through serial passages in linezolid sub-inhibitory concentrations. Combinations of linezolid with imipenem or anti-inflammatory agents were investigated using time-kill and modified checkerboard assays. Three Staphylococcus haemolyticus isolates (1.3%) from 2015 to 2016 were linezolid-resistant. One isolate carried cfr which was plasmid-borne, and together with another isolate which had a G2603T point mutation in the V domain of 23S rRNA gene. Successive exposure to linezolid sub-inhibitory concentrations was selected for three resistant Staphylococcus aureus mutants out of ten susceptible isolates. These mutants were more resistant towards different antibiotic classes than their susceptible parents. Linezolid combinations with imipenem, ibuprofen, or aspirin were synergistic against the isolates and mutants. Despite unregulated use of linezolid, resistance remains fairly low among the Egyptian isolates. Strict antimicrobial stewardship guidelines are needed in hospitals and the community to guard against further evolution of resistant mutants.

Linezolid-Resistant Staphylococcus haemolyticus: Emergence of G2447U and C2534U Mutations at the Domain V of 23Sr RNA Gene

Background: Linezolid an oxazolidinone drug available in both parenteral and oral formulations has emerged as a novel alternative to vancomycin and other second-generation drugs for the treatment of infections from gram-positive cocci. Clinical isolates of linezolid-resistant staphylococci and enterococci were reported. Since then, linezolid-resistant strains have become an increasing problem worldwide. The most frequently reported mechanisms of linezolid resistance include the mutation in 23S ribosomal nucleic acid and presence of cfr gene. Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) and vancomycin-resistant Enterococcus (VRE) have become a worrisome clinical problem. Objective: Therefore, we aimed to determine the distribution of linezolid-resistant strains in an inpatient setting of a tertiary-care hospital in India and to evaluate the resistance mechanisms among these isolates. In addition, the clonal diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE). Methods: The distribution, clonal diversity, and resistance mechanism of linezolid resistant-Staphylococcus haemolyticus (LRSH) strains were determined. The isolates were identified by MALDI-TOF. The mechanism of resistance was determined by sequence analysis of the domain V of 23SrRNA and screening for cfr gene. Clonal relatedness was defined by PFGE. Results: In total, 13 LRSH isolates were recovered from pus specimens. The 13 LRSH strains isolated had an linezolid MIC of 256 g/mL. Sequencing results revealed G2576T mutations in 7 (53.8%), G2447U in 4 (30.7%) and C2534U in 1 (7.6%) isolate of S. haemolyticus. One isolate of S. haemolyticus showed 2 simultaneous mutations (G2576T and G2447U) in the domain V region of 23Sr RNA gene. PFGE of the LR-SH isolates revealed the presence of 11 clones. Of the 11 clones, clones I and II had 2 isolates each. Isolates of clone I exhibited a band pattern identical with the previous isolates of LRSH isolated from the orthopedic unit. Similarly, isolates of clone II also shared the same band pattern with the previous LRSH isolates from the dermatology unit of our center. Conclusions: This study highlights the importance of continuous monitoring of vigilance of linezolid resistance in staphylococci. Rationalizing the use of linezolid and implementing methods to control the spread of hospital clones is of paramount importance to prevent further dissemination of these strains.Funding: NoneDisclosures: None

Research progress on the oxazolidinone drug linezolid resistance.

The oxazolidinone drug linezolid is mainly used for severe infections caused by multidrug-resistant Gram-positive bacteria. However, emerging linezolid resistance is aggravating difficulties in the treatment of certain infectious diseases. The objective of this review was to provide a reference for researchers and clinicians to be able to better face together the serious challenge of antimicrobial resistance. A systematic literature search was performed using PubMed, Web of Science, Google Scholar, and the China National Knowledge Infrastructure (CNKI) database. The articles were scrutinized to extract information on oxazolidinone drug linezolid resistance, and the prevalence of the resistance gene optrA. We reviewed the latest advances in epidemic properties, resistance mechanism, and transfer mechanism of linezolid resistance genes in different isolates isolated from various samples worldwide. Initially, it was thought that linezolid resistance was related to the change in drug target mediated by mutations in the 23S rRNA gene, rplC, rplD, and cfr. optrA was discovered in 2015, and is a gene encoding oxazolidinone resistance, which exists in both plasmids and chromosomes, but mostly plasmids. The emergence of the novel plasmid-borne ABC transporter gene optrA expanded the understanding of the mechanism of linezolid resistance. At present, the prevalence of linezolid resistance has become increasingly serious. The resistance gene optrA has been reported in Enterococcus, Staphylococcus squirrel and Streptococcus, which indicates that this gene has a strong ability to spread across bacteria, so the prevalence and spread of optrA gene should be monitored carefully.