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.

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.

Nasotracheal enterococcal carriage and resistomes: detection of optrA-, poxtA- and cfrD-carrying strains in migratory birds, livestock, pets, and in-contact humans in Spain.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

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.

Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the most difficult infections we have to treat. Linezolid is one of the effective treatment options for refractory MRSA infections. There are cases where we are forced to use long-term linezolid treatment for refractory MRSA infections. OBJECTIVE: To discuss the evolution of Linezolid resistance factors in clinical isolates of MRSA. METHODS: We investigated 16 MRSA isolated from a patient treated with linezolid for a long period of 75 days. We performed antibiotic susceptibility test, 23S rRNA genes sequencing analysis, Pulsed-field gel electrophoresis. RESULTS: MRSA isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment. The 23S rRNA sequencing analysis of linezolid-resistant strains that appeared 17 days after the start of treatment with linezolid revealed that all resistant MRSA had the G2576T substitution (Escherichia coli 23S rRNA gene number). The number of copies of this mutation increased with the use of linezolid. CONCLUSION: Long-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.

High occurrence of Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae harbouring oxazolidinone resistance genes in raw meat-based diets for companion animals - a public health issue, Switzerland, September 2018 to May 2020.

IntroductionEnterococci harbouring genes encoding resistance to florfenicol and the oxazolidinone antimicrobial linezolid have emerged among food-producing animals and meat thereof, but few studies have analysed their occurrence in raw meat-based diets (RMBDs) for pets.AimWe aimed to examine how far RMBDs may represent a source of bacteria with oxazolidinone resistance genes.MethodsFifty-nine samples of different types of RMBDs from 10 suppliers (three based in Germany, seven in Switzerland) were screened for florfenicol-resistant Gram-positive bacteria using a selective culture medium. Isolates were phenotypically and genotypically characterised.ResultsA total of 27 Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae isolates were obtained from 24 of the 59 samples. The optrA, poxtA, and cfr genes were identified in 24/27, 6/27 and 5/27 isolates, respectively. Chloramphenicol and linezolid minimum inhibitory concentrations (MICs) ranged from 24.0 mg/L-256.0 mg/L, and 1.5 mg/L-8.0 mg/L, respectively. According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, 26 of 27 isolates were resistant to chloramphenicol (MICs ≥ 32 mg/L), and two were resistant to linezolid (MICs ≥ 8 mg/L). Multilocus sequence typing analysis of the 17 E. faecalis isolates identified 10 different sequence types (ST)s, with ST593 (n = 4 isolates) and ST207 (n = 2 isolates) occurring more than once, and two novel STs (n = 2 isolates). E. faecium isolates belonged to four different STs (168, 264, 822, and 1846).ConclusionThe high occurrence in our sample of Gram-positive bacteria harbouring genes encoding resistance to the critical antimicrobial linezolid is of concern since such bacteria may spread from companion animals to humans upon close contact between pets and their owners.

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.

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

PURPOSE: The 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. METHODS: S. 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. RESULTS: The 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. CONCLUSIONS: The 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- and Multidrug-Resistant Enterococci in Raw Commercial Dog Food, Europe, 2019-2020.

We describe enterococci in raw-frozen dog food commercialized in Europe as a source of genes encoding resistance to the antibiotic drug linezolid and of strains and plasmids enriched in antibiotic-resistance and virulence genes in hospitalized patients. Whole-genome sequencing was fundamental to linking isolates from dog food to human cases across Europe.

Genetic Context of optrA and poxtA in Florfenicol-Resistant Enterococci Isolated from Flowing Surface Water in Switzerland.

Linezolid is an important last-resort antibiotic for the treatment of multidrug-resistant enterococci. The aim of this study was to further characterize the genetic context of optrA and poxtA in 10 florfenicol-resistant enterococci isolated from flowing surface water. In most genomes, optrA and poxtA were embedded in transposition units integrated into plasmids or into the chromosomal radC. For the first time, a chromosomally integrated optrA in an Enterococcus raffinosus isolate is described.

Emergence of linezolid-resistance in vancomycin-resistant Enterococcus faecium ST117 associated with increased linezolid-consumption.

OBJECTIVE: We aim to describe the epidemiological, clinical and microbiological characteristics of the linezolid- and vancomycin- resistant Enterococcus faecium (LVRE) in a tertiary care hospital in Germany. METHODS: We conducted a retrospective analysis of 196 LVRE cases observed from 1st January 2012 to 31th December 2018. Patients' medical charts were reviewed and available LVRE (n = 102) were subjected to whole-genome-sequencing. Antibiotic consumption was measured in defined daily dose (DDD)/100 bed-days (BD). RESULTS: The prevalence of LVRE isolates among VRE was 6.3 % in 2018. Most patients had an onco-hematological disease (134/196, 68.4 %). From 2012-2018 an increase of +356.7 % of linezolid defined daily dose/100 bed-days was observed. In 71.4 % (90/126, 70 missing values) of the patients, linezolid was prescribed in the previous 6 months. The median exposure to linezolid was 15 days (Interquartile, IQR 9-23). 42/196 (21.4 %) patients had an LVRE-related infection with an overall 30-day mortality rate of 33 %. In 121/196 (61.7 %) patients, linezolid-susceptible VREfm were isolated before LVRE, suggesting secondary acquisition of linezolid resistance. Genetic analysis revealed that most isolates belonged to ST117 (64/102 available isolates, 62.7 %). The G2576T 23S rDNA mutation was identified as the most common resistance mechanism (96/102, 94.1 %). poxtA was identified in two isolates, while cfr, and optrA were not detected. CONCLUSIONS: Incidence of LVRE related to 23S rDNA mutations is rising and probably associated with antibiotic consumption. Restrictions in the use of linezolid may be needed in order to retain therapeutic options in VRE.

Linezolid Resistance Genes in Enterococci Isolated from Sediment and Zooplankton in Two Italian Coastal Areas.

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs.IMPORTANCE Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria; therefore, the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA, and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples from two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environments could reflect their spillover from human and/or animal reservoirs and could indicate that coastal seawaters also might represent a source of these resistance genes.

Genomic Information on Linezolid-Resistant Sequence-Type 398 Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolated from a Pig.

The frequent occurrence of sequence-type 398 (ST398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs has become a major public health concern owing to the increased zoonotic potential of the pathogen. Recently, a novel oxazolidinone resistance gene, chloramphenicol-florfenicol resistant (cfr), conferring multiresistance phenotypes to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA), has been found among ST398 LA-MRSA strains isolated from pigs. In this study, we report the first in silico genome analysis of a linezolid-resistant ST398 LA-MRSA strain, designated PJFA-521M, recovered from a pig in Korea. Genomic analyses revealed that the presence of the cfr gene was responsible for the observed linezolid resistance in the PJFA-521M strain. Moreover, newer antimicrobial resistance genes, such as the dfrG, aadE, spw, lsa(E), lnu(B), and fexA genes, were found in the PJFA-521M strain. In addition to the genetic elements for antimicrobial resistance, the carriage of various virulence genes for adherence, invasion, and immunomodulation was identified in the genome, especially within several mobile genetic elements (MGEs). The presence of multiple antimicrobial resistance genes and virulence genes on MGEs in the genome of a linezolid-resistant ST398 LA-MRSA should raise awareness regarding the use of other antimicrobial agents in pig farms and may also provide selective pressure for the prevalence of the cfr gene and the associated multidrug-resistant phenotype.

Molecular characteristics and predictors of mortality among Gram-positive bacteria isolated from bloodstream infections in critically ill patients during a 5-year period (2012-2016).

To identify the molecular characteristics of Gram-positive cocci isolated from blood cultures and clinical outcome among critically ill patients. This retrospective study was conducted in the general intensive care unit of the University General Hospital of Patras, Greece, during a 5-year period (2012-2016). All adult patients with a Gram-positive BSI were included. PCR was applied to identify mecA gene (staphylococci); vanA, vanB, and vanC genes (enterococci). Linezolid-resistant S. epidermidis, MRSA, and VRE were further typed by multilocus sequence typing. Mutations in region V of 23S rDNA and ribosomal protein L4were investigated by PCR and sequencing analysis. The presence of the cfr gene was tested by PCR. In total, 141 Gram-positive BSIs were included. Coagulase-negative staphylococci predominated (n = 69; 65 methicillin-resistant, 23 linezolid-resistant carrying both C2534T and T2504A mutations and belonging to the ST22 clone), followed by enterococci (n = 46; 11 vancomycin-resistant carrying vanA gene, classified into four clones), S. aureus (n = 22; 10 methicillin-resistant, classified into three clones) and streptococci (n = 4). The most common type of infection was catheter-related (66; 46.8%), followed by primary BSI (28; 19.9%). Overall 14-day fatality was 24.8%. Multivariate analysis revealed septic shock as independent predictor of fatality, while appropriate empiric antimicrobial treatment and catheter-related BSI were identified as a predictor of good prognosis. Even though most of Gram-positive cocci were multidrug-resistant, fatality rate was low, associated with catheter-related BSIs. Among CNS, LR isolates represented one-third of BSIs due to the dissemination of ST22 S. epidermidis propagated by utilization of linezolid.

Linezolid- and Vancomycin-resistant Enterococcus faecium in Solid Organ Transplant Recipients: Infection Control and Antimicrobial Stewardship Using Whole Genome Sequencing.

BACKGROUND: Vancomycin-resistant enterococci are an important cause of healthcare-associated infections and are inherently resistant to many commonly used antibiotics. Linezolid is the only drug currently approved by the US Food and Drug Administration to treat vancomycin-resistant enterococci; however, resistance to this antibiotic appears to be increasing. Although outbreaks of linezolid- and vancomycin-resistant Enterococcus faecium (LR-VRE) in solid organ transplant recipients remain uncommon, they represent a major challenge for infection control and hospital epidemiology. METHODS: We describe a cluster of 4 LR-VRE infections among a group of liver and multivisceral transplant recipients in a single intensive care unit. Failure of treatment with linezolid in 2 cases led to a review of standard clinical laboratory methods for susceptibility determination. Testing by alternative methods including whole genome sequencing (WGS) and a comprehensive outbreak investigation including sampling of staff members and surfaces was performed. RESULTS: Review of laboratory testing methods revealed a limitation in the VITEK 2 system with regard to reporting resistance to linezolid. Linezolid resistance in all cases was confirmed by E-test method. The use of WGS identified a resistant subpopulation with the G2376C mutation in the 23S ribosomal RNA. Sampling of staff members' dominant hands as well as sampling of surfaces in the unit identified no contaminated sources for transmission. CONCLUSIONS: This cluster of LR-VRE in transplant recipients highlights the possible shortcomings of standard microbiology laboratory methods and underscores the importance of WGS to identify resistance mechanisms that can inform patient care, as well as infection control and antibiotic stewardship measures.

Molecular characteristics of oxazolidinone resistance in enterococci from a multicenter study in China.

BACKGROUND: Linezolid-resistant enterococci pose great challenges in clinical practice. The aim of this study is to study the mechanisms underlying the resistance and genetic environment of antimicrobial resistance gene of linezolid-resistant enterococci. RESULTS: The linezolid MICs of 16 enterococci were 4 mg/L to 16 mg/L. Four strains belonged to multi-drug resistant (MDR) bacteria. The sequence types (STs) of 13 enterococci strains performed WGS were diverse: 3 ST476, 1 ST86, ST116, ST480, ST59, ST416, ST21, ST67, ST16, ST585 and ST18. None of them carried multi-drug resistance gene cfr. Only one strain had the G2658 T mutation of target 23S rRNA gene. Thirteen (13/16, 81.3%) strains harbored the novel oxazolidinone resistance gene optrA. WGS analysis showed that the optrA gene was flanked by sequence IS1216E insertion in 13 strains, and optrA was adjacent to transposons Tn558 in two strains and Tn554 in one strain. The optrA gene was identified to be co-localized with fexA, the resistance genes mediated florfenicol resistance in 13 strains, and ermA1, the resistance genes mediated erythromycin resistance in 9 strains, indicating that linezolid-resistant strains may be selected due to non-oxazolidinone antibiotics (i.e. macrolides and florfenicol) usage. CONCLUSION: Our findings demonstrate the high diversity of optrA-carrying genetic platforms. The mobile genetic elements (MGEs) may play an important role in the dissemination of optrA into the enterococci isolates of human origin. The genetic evidence of transferable feature and co-selection of optrA should be gave more attention in clinical practice.

Validating a screening agar for linezolid-resistant enterococci.

BACKGROUND: Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. Some countries report an increasing number of isolates with resistance to linezolid. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). However, a suitable selective medium or a genetic test is not available. Our aim was to establish a selective screening agar for LRE detection and validate its application with a comprehensive collection of clinical LRE and linezolid-susceptible enterococci. METHODS: We decided to combine the selective power of an enterococcal screening agar with a supplementation of linezolid. Several rounds of analyses with reference, control and test strains and under varying linezolid concentrations of a wider and a smaller range were investigated and assessed. The collection of linezolid-resistant enterococcal control strains included isolates with different resistance mechanisms (23S rDNA mutations, cfr(B), optrA, poxtA). Finally, we validated our LRE screening agar with 400 samples sent to our National Reference Centre in 2019. RESULTS: Several rounds of pre-tests and confirmatory analyses favored Enterococcosel® Agar supplemented with a concentration of 2 mg/L linezolid. A 48 h incubation period was essential for accurate identification of LRE strains. Performance of the LRE screening agar revealed a sensitivity of 96.6% and a specificity of 94.4%. CONCLUSIONS: Here we describe preparation of a suitable screening agar and a procedure to identify LRE isolates with high accuracy.

Linezolid resistant coagulase negative staphylococci (LRCoNS) with novel mutations causing blood stream infections (BSI) in India.

BACKGROUND: Coagulase-negative Staphylococci (CoNS) have emerged as a major causative agent of blood-stream infections (BSI). Linezolid (LZD) is currently used for treating glycopeptide and methicillin-resistant staphylococci. It is important to understand the resistance mechanism and probable transmission of LZD resistant (LR) CoNS within the hospital. METHODS: Clinically significant LRCoNS from patients with BSI were characterized using MALDI-TOF and 16S rRNA gene sequence analysis. Antimicrobial susceptibility and MIC of vancomycin and LZD were determined. LZD resistance mechanisms using PCR for the cfr gene and mutation in the V domain of the 23S rRNA gene were studied. RESULTS: The MIC of LZD ranged from 8 to 32 µg/ml. LR was observed in three different CoNS species from diverse locations within the hospital. The cfr gene was identified in all the isolates. Sequence analysis of V domain region of 23S rRNA gene confirmed mutation in single copy among 12/15 isolates with novel mutations: G2614 T and C2384T. All infections were nosocomially acquired and LZD resistance was emerging in the absence of prior LZD use. Horizontal spread of resistant isolates and cfr gene among diverse species were the probable mechanisms of transmission. CONCLUSION: The study highlights the novel mutations associated with LRCoNS and the importance of surveillance & transmission pathway within the hospital. It also systematically discusses the published information on LRCoNS.

Recommendations To Address the Difficulties Encountered When Determining Linezolid Resistance from Whole-Genome Sequencing Data.

Mutations associated with linezolid resistance within the V domain of 23S rRNA are annotated using an Escherichia coli numbering system. The 23S rRNA gene varies in length, nucleotide sequence, and copy number among bacterial species. Consequently, this numbering system is not intuitive and can lead to confusion when mutation sites are being located using whole-genome sequencing data. Using the mutation G2576T as an example, we demonstrate the difficulties associated with using the E. coli numbering system.

A high incidence and coexistence of multiresistance genes cfr and optrA among linezolid-resistant enterococci isolated from a teaching hospital in Wenzhou, China.

Linezolid is considered as a last-resort antimicrobial agent, the resistance of which is of great concern. The aim of this study was to investigate the mechanisms and transferability of linezolid resistance and molecular epidemiology of linezolid-resistant enterococcal isolates in Wenzhou, China. A collection of 1623 enterococcal strains, including 789 Enterococcus faecalis and 834 Enterococcus faecium, were isolated from our hospital during 2011-2016. Antimicrobial susceptibility testing and clinical data analysis were performed. Molecular mechanisms of linezolid resistance, including the existence of resistance genes cfr and optrA, as well as the mutations in 23S rRNA and ribosomal proteins L3, L4, and L22, were investigated by PCR and sequencing. Conjugation experiments were conducted, and epidemiological characteristics were analyzed by PFGE and MLST. In our study, 31 (3.93%) E. faecalis and 2 (0.24%) E. faecium exhibited resistance to linezolid. Risk factors correlated with linezolid-resistant enterococcal infections included gastrointestinal surgery hospitalization, urogenital disorders, tumor, diabetes, and polymicrobial infections. Among these isolates, 6 (18.18%) harbored cfr, 9 (27.27%) harbored optrA, and 18 (54.55%) co-harbored cfr and optrA. However, mutational mechanisms were not found in this study. Conjugation experiments demonstrated the transferability of cfr and optrA between Gram-positive and Gram-negative bacteria. The clone of these isolates was diverse and scattered. It is noteworthy that cfr and optrA were the main mechanisms of linezolid resistance in this study, posing a potential risk of spread of linezolid resistance. Strikingly, it reported firstly that the two transferable resistance genes cfr and optrA coexisted in the same E. faecalis isolates.