Phylogeny of Transferable Oxazolidinone Resistance Genes and Homologs.

Oxazolidinone resistance, especially transmissible resistance, is a major public health concern, and the origin of this resistance mechanism is not yet resolved. This study aims to delve into the phylogenetic origin of the transmissible oxazolidinone resistance mechanisms conferring cross-resistance to other drugs of human and veterinary importance. The amino acid sequences of the five cfr ribosomal methylases and optrA and poxtA were used as queries in searches against 219,549 bacterial proteomes in the NCBI RefSeq database. Hits with >40% amino acid identity and >80% query coverage were aligned, and phylogenetic trees were reconstructed. All five cfr genes yielded highly similar trees, with rlmN housekeeping ribosomal methylases located basal to the sister groups of S-adenosyl-methionine-dependent methyltransferases from various Deltaproteobacteria and Actinomycetia, including antibiotic-producing Streptomyces species, and the monophyletic group of cfr genes. The basal branches of the latter contained paenibacilli and other soil bacteria; they then could be split into the clades [cfr(C):cfr(E)] and [[cfr:cfr(B)]:cfr(D)], always with different Bacillaceae in their stems. Lachnospiraceae were encountered in the basal branches of both optrA and poxtA trees. The ultimate origin of the cfr genes is the rlmN housekeeping ribosomal methylases, which evolved into a suicide-avoiding methylase in antibiotic producers; a soil organism (Lachnospiraceae, Paenibacilli) probably acted as a transfer organism into pathogenic bacteria. In the case of optrA, the porcine pathogenic Streptococcus suis was present in all branches, while the proteins closest to poxtA originated from Clostridia.

Mapping the widespread distribution and transmission dynamics of linezolid resistance in humans, animals, and the environment.

BACKGROUND: The rise of linezolid resistance has been widely observed both in clinical and non-clinical settings. However, there were still data gaps regarding the comprehensive prevalence and interconnections of linezolid resistance genes across various niches. RESULTS: We screened for potential linezolid resistance gene reservoirs in the intestines of both humans and animals, in meat samples, as well as in water sources. A total of 796 bacteria strains out of 1538 non-duplicated samples were identified to be positive for at least one linezolid resistance gene, optrA, poxtA, cfr, and cfr(D). The prevalence of optrA reached 100% (95% CI 96.3-100%) in the intestines of pigs, followed by fish, ducks, and chicken at 77.5% (95% CI 67.2-85.3%), 62.0% (95% CI 52.2-70.9%), and 61.0% (95% CI 51.2-70.0%), respectively. The meat and water samples presented prevalences of 80.0% (95% CI 70.6-87.0%) and 38.0% (95% CI 25.9-51.9%), respectively. The unreported prevalence of the cfr(D) gene was also relatively higher at 13.0% (95% CI 7.8-21.0%) and 19.0% (95% CI 10.9-25.6%) for the feces samples of ducks and pigs, respectively. Enterococci were the predominant hosts for all genes, while several non-enterococcal species were also identified. Phylogenetic analysis revealed a significant genetic distance among linezolid resistance gene reservoirs, with polyclonal structures observed in strains within the same niche. Similar genetic arrays harboring assorted insertion sequences or transposons were shared by reservoirs displaying heterogeneous backgrounds, though large diversity in the genetic environment of linezolid resistance genes was also observed. CONCLUSIONS: The linezolid resistance genes were widespread among various niches. The horizontal transfer played a crucial role in driving the circulation of linezolid resistance reservoirs at the human-animal-environment interfaces. Video Abstract.

Genetic Diversification and Resistome of Coagulase-Negative Staphylococci from Nostrils of Healthy Dogs and Dog-Owners in La Rioja, Spain.

Coagulase-negative staphylococci (CoNS) species in healthy dogs and their owners could be transferred between these hosts and carry diverse antimicrobial resistance (AMR) genes of public health concern. This study determined the frequency, diversity, and AMR genes of nasal CoNS from healthy dogs and in-contact people as well as the rate of intra-household (between healthy dogs and dog-owners) transmission of CoNS. Nasal samples were collected and processed from 34 dogs and 41 humans from 27 households, and CoNS identification was done by MALDI-TOF-MS. The AMR determinants and genetic lineages were determined by PCR/sequencing. A total of 216 CoNS isolates were initially obtained and identified, and the AMR phenotypes were determined. From these, 130 non-repetitive CoNS were selected (one isolate of each species per sample or more than one if they presented different AMR phenotypes) and further characterized. The predominant species from dog carriers were S. epidermidis (26.5%), S. hominis (8.8%), and S. cohnii (8.8%), whereas in the human carriers, the predominant ones were S. epidermidis (80.4%), S. lugdunensis (9.8%), and S. hominis (9.8%). Intra-host species diversity (>one CoNS species) was detected in 37.5% of dogs and 21.6% of dog-owners. Conversely, 50% of dogs and 70.3% of dog-owners had intra-species AMR diversity (2-4 AMR-CoNS profiles). About 20% were susceptible to all antimicrobial agents tested, 31.5% displayed a multidrug resistance phenotype, and 17.4% were mecA-positive, located in SCCmec type V (24.2%), III (18.1%), IVc (12.1%), and II (6.1%). The other mec-A positive CoNS isolates (39.5%) had non-typeable SCCmec. The highest AMR rates were found against erythromycin (32.3%/mph(C), msr(A)) and mupirocin (20.8%/mupA), but the resistance rates for other antimicrobial agents were <10% each. Remarkably, one linezolid-resistant S. epidermidis-ST35 isolate was identified and mediated by four amino acid substitutions in L3 and one in L4 ribosomal proteins. Dogs and dog-owners as carriers of S. epidermidis with similar AMR patterns and genetic lineages (ST59, ST61, ST166 and ST278) were detected in four households (14.8%). Diverse CoNS carriage and moderate level of AMR were obtained from this study. The detection of CoNS carrying diverse SCCmec elements and intra-species AMR diversity highlights the roles of dog ownership in the potential transmission of antimicrobial-resistant CoNS in either direction.

Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea.

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Linezolid Resistance Genes and Mutations among Linezolid-Susceptible Enterococcus spp.-A Loose Cannon?

The National Reference Centre for Enterococci receives an increasing number of linezolid-resistant Enterococcus isolates. Linezolid (LIN) resistance is mediated by G2576T 23S rDNA gene mutations and/or acquisition of resistance genes (cfr, optrA, poxtA). There are anecdotal reports that those resistance traits may be present in phenotypically linezolid-susceptible isolates. We aimed to determine the prevalence of LIN resistance genes and mutations in enterococci with a LIN MIC of 4 mg/L in broth microdilution (EUCAST = susceptible) isolated from German hospital patients 2019-2021. LIN MICs were additionally determined by ETEST® and VITEK2. Selected strains were subjected to LIN selective pressure and growth was monitored with increasing antibiotic concentrations. We received 195 isolates (LIN MIC = 4 mg/L). In total, 78/195 (40%) isolates contained either a putative resistance gene, the G2576T mutation, or a combination thereof. Very major error was high for broth microdilution. The ability to predict phenotypic resistance from genotypic profile was highest for G2576T-mediated resistance. Selection experiments revealed that, in particular, E. faecium isolates with resistance gene mutations or poxtA rapidly adapt to MICs above the clinical breakpoint. In conclusion, LIN resistance genes and mutations can be observed in phenotypically linezolid-susceptible enterococci. Those isolates may rapidly develop resistance under LIN selective pressure potentially leading to treatment failure.

Evaluation of different linezolid susceptibility testing methods and detection of linezolid resistance gene (cfr) in staphylococcal isolates.

Linezolid is an effective oxazolidinone antibiotic against multi resistant Gram-positive organisms. Linezolid resistance is an emerging problem and some controversy exists about the reliability of different phenotypic methods of linezolid susceptibility testing. Fifty isolates each of methicillin resistant S. aureus (MRSA) and Staphylococcus haemolyticus were tested for linezolid susceptibility using Kirby-Bauer disc diffusion, E-test, automated system VITEK2, Broth micro-dilution (reference method) and PCR for the cfr gene. Six resistant isolates were identified, three each in MRSA and S. haemolyticus, all carrying the cfr gene. E-test and VITEK2 were found to be more accurate than disc diffusion test.

Risk exploration and prediction model construction for linezolid-resistant Enterococcus faecalis based on big data in a province in southern China.

BACKGROUND: Enterococcus faecalis is a common cause of healthcare-associated infections. Its resistance to linezolid, the antibiotic of last resort for vancomycin-resistant enterococci, has become a growing threat in healthcare settings. METHODS: We analyzed the data of E. faecalis isolates from 26 medical institutions between 2018 and 2020 and performed univariate and multivariate logistic regression analyses to determine the independent predictors for linezolid-resistant E. faecalis (LREFs). Then, we used the artificial neural network (ANN) and logistic regression (LR) to build a prediction model for linezolid resistance and performed a performance evaluation and comparison. RESULTS: Of 12,089 E. faecalis strains, 755 (6.25%) were resistant to linezolid. Among vancomycin-resistant E. faecalis, the linezolid-resistant rate was 24.44%, higher than that of vancomycin-susceptible E. faecalis (p < 0.0001). Univariate and multivariate regression analyses showed that gender, age, specimen type, length of stay before culture, season, region, GDP (gross domestic product), number of beds, and hospital level were predictors of linezolid resistance. Both the ANN and LR models constructed in the study performed well in predicting linezolid resistance in E. faecalis, with AUCs of 0.754 and 0.741 in the validation set, respectively. However, synthetic minority oversampling technique (SMOTE) did not improve the prediction ability of the models. CONCLUSION: E. faecalis linezolid-resistant rates varied by specimen site, geographic region, GDP level, facility level, and the number of beds. At the same time, community-acquired E. faecalis with linezolid resistance should be monitored closely. We can use the prediction model to guide clinical medication and take timely prevention and control measures.

Recombination-mediated dissemination of Methicillin-resistant S. aureus clonal complex 1 in the Egyptian health care settings.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly evolving pathogen that is frequently associated with outbreaks and sustained epidemics. This study investigated the population structure, resistome, virulome, and the correlation between antimicrobial resistance determinants with phenotypic resistance profiles of 36 representative hospital-acquired MRSA isolates recovered from hospital settings in Egypt. RESULTS: The community-acquired MRSA lineage, clonal complex 1 (CC1) was the most frequently detected clone, followed by three other globally disseminated clones, CC121, CC8, and CC22. Most isolates carried SCCmec type V and more than half of isolates demonstrated multi-drug resistant phenotypes. Resistance to linezolid, a last resort antibiotic for treating multidrug resistant MRSA, was observed in 11.11% of the isolates belonging to different genetic backgrounds. Virulome analysis indicated that most isolates harboured a large pool of virulence factors and toxins. Genes encoding aureolysin, gamma hemolysins, and serine proteases were the most frequently detected virulence encoding genes. CC1 was observed to have a high pool of AMR resistance determinants including cfr, qacA, and qacB genes, which are involved in linezolid and quaternary ammonium compounds resistance, as well as high content of virulence-related genes, including both of the PVL toxin genes. Molecular clock analysis revealed that CC1 had the greatest frequency of recombination (compared to mutation) among the four major clones, supporting the role of horizontal gene transfer in modulating AMR and hypervirulence in this clone. CONCLUSIONS: This pilot study provided evidence on the dissemination success of CA-MRSA clone CC1 among Egyptian hospitals. Co-detection of multiple AMR and virulence genes in this lineage pose a broad public health risk, with implications for successful treatment. The results of this study, together with other surveillance studies in Egypt, should be used to develop strategies for controlling MRSA infections in Egyptian health-care settings.

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.

The Synergistic Activity of Rhamnolipid Combined with Linezolid against Linezolid-Resistant Enterococcus faecium.

Enterococci resistance is increasing sharply, which poses a serious threat to public health. Rhamnolipids are a kind of amphiphilic compound used for its bioactivities, while the combination of nontraditional drugs to restore linezolid activity is an attractive strategy to treat infections caused by these pathogens. This study aimed to investigate the activity of linezolid in combination with the rhamnolipids against Enterococcus faecium. Here, we determined that the rhamnolipids could enhance the efficacy of linezolid against enterococci infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, an anti-biofilm assay, molecular simulation dynamics, and mouse infection models. We identified that the combination of rhamnolipids and linezolid restored the linezolid sensitivity. Anti-biofilm experiments show that our new scheme can effectively inhibit biofilm generation. The mouse infection model demonstrated that the combination therapy significantly reduced the bacterial load in the feces, colons, and kidneys following subcutaneous administration. This study showed that rhamnolipids could play a synergistic role with linezolid against Enterococcus. Our combined agents could be appealing candidates for developing new combinatorial agents to restore antibiotic efficacy in the treatment of linezolid-resistant Enterococcus infections.

Within-Host Diversity of Coagulase-Negative Staphylococci Resistome from Healthy Pigs and Pig Farmers, with the Detection of cfr-Carrying Strains and MDR-S. borealis.

The ecology and diversity of resistome in coagulase-negative staphylococci (CoNS) from healthy pigs and pig farmers are rarely available as most studies focused on the livestock-associated methicillin-resistant S. aureus. This study aims to characterize the antimicrobial resistance (AMR) mechanisms, intra-host species diversity (more than one species in a host), and intra-species AMR diversity (same species with more than one AMR profile) in CoNS recovered from the nasal cavities of healthy pigs and pig farmers. One-hundred-and-one CoNS strains previously recovered from 40 pigs and 10 pig farmers from four Spanish pig farms were tested to determine their AMR profiles. Non-repetitive strains were selected (n = 75) and their AMR genes, SCCmec types, and genetic lineages were analyzed by PCR/sequencing. Of the non-repetitive strains, 92% showed a multidrug resistance (MDR) phenotype, and 52% were mecA-positive, which were associated with SCCmec types V (46.2%), IVb (20.5%), and IVc (5.1%). A total of 28% of the pigs and pig farmers had intra-host species diversity, while 26% had intra-species AMR diversity. High repertoires of AMR genes were detected, including unusual ones such as tetO, ermT, erm43, and cfr. Most important was the detection of cfr (in S. saprophyticus and S. epidermidis-ST16) in pigs and pig farmers; whereas MDR-S. borealis strains were identified in pig farmers. Pig-to-pig transmission of CoNS with similar AMR genes and SCCmec types was detected in 42.5% of pigs. The high level of multidrug, within-host, and intra-species resistome diversity in the nasal CoNS highlights their ability to be AMR gene reservoirs in healthy pigs and pig farmers. The detection of MDR-S. borealis and linezolid-resistant strains underscore the need for comprehensive and continuous surveillance of MDR-CoNS at the pig farm level.

Rapid culture-based LNZ test for detection of linezolid susceptibility/resistance in staphylococci and enterococci.

A rapid, easy-to-handle, cost-effective and universal culture-based test was developed for the identification of linezolid resistance among the most clinically relevant enterococcal and staphylococcal species. Our technique was tested using linezolid-resistant (n = 50) and linezolid-susceptible (n = 67) Gram-positive isolates: 34 Enterococcus faecium, 20 Enterococcus faecalis, 20 Staphylococcus aureus, 38 Staphylococcus epidermidis, and 5 Staphylococcus capitis. The susceptibility/resistance phenotype of E. faecium, E. faecalis, S. aureus, and S. epidermidis to linezolid was detected within 4.5 hours, while an extended timeframe was actually required for S. capitis (6.5 hours). The Rapid LNZ test showed a full agreement with the standard broth microdilution method, independently of the molecular resistance mechanism and MIC values, with sensitivities and specificities of 100% for all species.

Molecular Characterization of Clinical Linezolid-Resistant Staphylococcus epidermidis in a Tertiary Care Hospital.

Staphylococcus epidermidis (S. epidermidis) is part of the human skin flora but can also cause nosocomial infections, such as device-associated infections, especially in vulnerable patient groups. Here, we investigated clinical isolates of linezolid-resistant S. epidermidis (LRSE) collected from blood cultures at the University Hospital Münster (UHM) during the period 2020-2022. All detected isolates were subjected to whole genome sequencing (WGS) and the relatedness of the isolates was determined using core genome multilocus sequence typing (cgMLST). The 15 LRSE isolates detected were classified as multilocus sequence type (ST) 2 carrying the staphylococcal cassette chromosome mec (SCCmec) type III. All isolates showed high-level resistance for linezolid by gradient tests. However, no isolate carried the cfr gene that is often associated with linezolid resistance. Analysis of cgMLST data sets revealed a cluster of six closely related LRSE isolates, suggesting a transmission event on a hematological/oncological ward at our hospital. Among the included patients, the majority of patients affected by LRSE infections had underlying hematological malignancies. This confirms previous observations that this patient group is particularly vulnerable to LRSE infection. Our data emphasize that the surveillance of LRSE in the hospital setting is a necessary step to prevent the spread of multidrug-resistant S. epidermidis among vulnerable patient groups, such as patients with hematological malignancies, immunosuppression or patients in intensive care units.

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.

Detection of a Novel G2603T Mutation in cfr Harboring Linezolid-Resistant Staphylococcus haemolyticus : First Report from India.

Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial pathogen. Linezolid is useful in the treatment of severe infections caused by methicillin-resistant Staphylococci . Resistance to linezolid in Staphylococci is due to one or more of the following mechanisms: acquisition of the cfr (chloramphenicol florfenicol resistance) gene, mutation in the central loop of domain V of the 23S rRNA, and mutation in the rplC and rplD genes. This study was carried out to detect and characterize resistance to linezolid among the clinical isolates of Staphylococcus haemolyticus . Materials and Methods The study included 84 clinical isolates of Staphylococcus haemolyticus . Susceptibility to various antibiotics was determined by disc diffusion method. Minimum inhibitory concentration (MIC) was determined by agar dilution method for linezolid. Methicillin resistance was screened using oxacillin and cefoxitin disc. Polymerase chain reaction was done to detect mecA, cfr and mutations in the V domain of the 23S rRNA gene. Results Resistance to linezolid was exhibited by 3 of the 84 study isolates with MIC more than 128 µg/mL. The cfr gene was detected in all the three isolates. The G2603T mutation was observed in the domain V of the 23S rRNA among two isolates, whereas one isolate lacked any mutation. Conclusion The emergence and spread of linezolid-resistant Staphylococcus haemolyticus isolates carrying G2603T mutation in the domain V of the 23S rRNA and harboring the cfr gene pose a threat in clinical practice.

Prevalence and Associated Factors of optrA-Positive-Enterococcus faecalis in Different Reservoirs around Farms in Vietnam.

Linezolid is an antibiotic of last resort for the treatment of infections caused by Gram-positive bacteria, including vancomycin-resistant enterococci. Enterococcus faecalis, a member of enterococci, is a significant pathogen in nosocomial infections. E. faecalis resistance to linezolid is frequently related to the presence of optrA, which is often co-carried with fex, phenicol exporter genes, and erm genes encoding macrolide resistance. Therefore, the common use of antibiotics in veterinary might promote the occurrence of optrA in livestock settings. This is a cross-sectional study aiming to investigate the prevalence of optrA positive E. faecalis (OPEfs) in 6 reservoirs in farms in Ha Nam province, Vietnam, and its associated factors and to explore genetic relationships of OPEfs isolates. Among 639 collected samples, the prevalence of OPEfs was highest in flies, 46.8% (51/109), followed by chickens 37.3% (72/193), dogs 33.3% (17/51), humans 18.7% (26/139), wastewater 16.4% (11/67) and pigs 11.3%, (14/80). The total feeding area and total livestock unit of the farm were associated with the presence of OPEfs in chickens, flies, and wastewater. Among 186 OPEfs strains, 86% were resistant to linezolid. The presence of optrA was also related to the resistant phenotype against linezolid and levofloxacin of E. faecalis isolates. Close genotypic relationships identified by Pulsed Field Gel Electrophoresis between OPEfs isolates recovered from flies and other reservoirs including chickens, pigs, dogs, and wastewater suggested the role of flies in the transmission of antibiotic-resistant pathogens. These results provided warnings of linezolid resistance although it is not used in livestock.

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.

Complete genome sequence of cfr(B)-carrying Enterococcus raffinosus isolated from bile in a patient in Japan.

OBJECTIVES: Linezolid is an antibiotic used to treat infectious diseases caused by vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Recently, Enterococcus Spp.-carrying mobile linezolid resistance genes were reported. Herein, we report the complete genome sequence of Enterococcus raffinosus JARB-HU0741, which was isolated from a bile sample of a patient in Japan on May 5, 2021, and carries a linezolid resistance gene, cfr(B). Nevertheless, this isolate was susceptible to linezolid. METHODS: Whole-genome sequencing was performed using HiSeq X FIVE (Illumina) and GridION (Oxford Nanopore Technologies). The sequence reads were assembled using Unicycler v0.4.8, and the complete genome was annotated using DFAST v1.2.18. Antimicrobial resistance genes were detected with Abricate v1.0.1, using the ResFinder database. The minimum inhibitory concentrations (MICs) were determined using broth microdilution and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: E. raffinosus JARB-HU0741 contained a 3 248 808-bp chromosome and a 1 156 277-bp megaplasmid. cfr(B) was present in the Tn6218-like transposon, which was inserted into a gene encoding a PRD domain-containing protein present in the megaplasmid, but the isolate was susceptible to linezolid (MIC, 0.5 µg/mL). The Tn6218-like transposon was similar to the Tn6218 of Clostridioides difficile Ox3196 and the Tn6218-like transposon of Enterococcus faecium UW11733; however, three genes encoding a topoisomerase, an S-adenosylmethionine-dependent methyltransferase, and a TetR family transcriptional regulator were present in the previous Tn6218- or Tn6218-like transposon. CONCLUSION: This is the first report of the complete genome sequence of E. raffinosus carrying cfr(B). E. raffinosus carrying cfr(B) without linezolid resistance poses a threat, as it could serve as a reservoir for mobile linezolid resistance genes.

Complete genome sequence of optrA-carrying Enterococcus faecalis isolated from open pus in a Japanese patient.

OBJECTIVES: The occurrence of linezolid resistance in enterococci has recently increased. Here, we report the genomic characterization of Enterococcus faecalis strain JARB-HU0796-isolated from the open pus of a patient in Hiroshima, Japan-which shows nonsusceptibility to linezolid (MIC of 4 µg/mL). METHODS: JARB-HU0796 whole-genome sequencing was performed using short-read sequencing with Illumina Hiseq X Five and long-read sequencing using GridION. These reads were collected using the assembly pipeline Unicycler and annotated with DFAST. Antimicrobial resistance genes were detected using the Abricate and ResFinder databases, and the sequence type identified using PubMLST. The antimicrobial susceptibility of JARB-HU0796 was determined with the Eiken dry-plate QH02 system. RESULTS: The JARB-HU0796 complete genome contained a circular chromosome (2 722 585 bp) and two circular plasmids (85 996 bp and 58 872 bp). The chromosome harbours the optrA gene, which confers resistance to oxazolidinones and phenicols. JARB-HU0796 showed nonsusceptibility to linezolid and multidrug resistance to other antibiotics. MLST analysis identified JARB-HU0796 as ST476, similar to the optrA-positive E. faecalis ST476 isolates from swine (South Korea, 2020) and pet food (Switzerland, 2022). The optrA region of JARB-HU0796 is nearly identical to that of ST476 E. faecalis strain TZ2, isolated from humans (China, 2013). CONCLUSIONS: To the best of our knowledge, this is the first report of the complete genome sequence of E. faecalis ST476 carrying optrA on a chromosome isolated from a patient in Japan. The strain may have originated in animals, suggesting that the organisms acquired resistance to linezolid because the optrA gene may be closely spread between animals and humans.