OBJECTIVES:This study aimed to investigate the "seesaw effect" of daptomycin (DAP) and ceftobiprole (BPR) on DAP-resistant (DAP-R) methicillin-resistant Staphylococcus aureus (MRSA) isolates. METHODS:Broth microdilution minimum inhibitory concentrations (MICs) of DAP and BPR were tested for laboratory-derived and clinical DAP-R MRSA isolates to estimate the "seesaw effect." Time-kill curves for seven representative DAP-R isolates were obtained using DAP and BPR to validate their synergistic activity in vitro. Whole genome sequencing as well as deletion and complementation of the mprF gene were performed to investigate the mechanisms of the "seesaw effect." RESULTS:The BPR MICs decreased by half-fold in DAP-R MRSA isolates. The synergistic effect of DAP and BPR against representative clinical and community-associated MRSA (CA-MRSA) isolates was demonstrated in time-kill analyses, showing that synergistic activity was preferred in CA-MRSA compared with hospital-associated MRSA. The mprF mutations were identified in isolates exhibiting the "seesaw effect." These mutations increased the DAP MIC while decreasing the BPR MIC. CONCLUSIONS:The "seesaw effect" between DAP and BPR was prevalent among DAP-R MRSA isolates. This phenomenon was associated with the mprF mutations of MRSA.

Objective This study aimed to investigate the impact of cold atmospheric-pressure plasma (CAP) on multidrug-resistant (MDR) bacteria on various surfaces under nosocomial circumstances and the underlying mechanism.Method Four common MDR bacteria (carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and carbapenem-resistant Klebsiella pneumoniae) were inoculated on nosocomial surfaces, which were subsequently exposed to CAP. Then the bacteria from surfaces were recovered and diluted. The killing curve was analyzed to evaluate the sterilization effects of CAP. Electron microscopy was used to evaluate the changes in cell morphology.Result In the CAP-producing device, most of the MDR bacteria were nearly inactivated after 2 h of CAP treatment. Under the simulated ward, CAP exerted an inactivating effect on MDR bacteria. Scanning electron microscopy revealed that the surface of MDR bacteria became blurred, the bodies ruptured and adhered to each other after CAP treatment. The cell walls were thinner as revealed by transmission electron microscopy.Conclusion CAP could inactivate the most common MDR bacteria on nosocomial surfaces in simulation ward settings by destroying the structure of pathogens. Our data provided insights into the sterilization of MDR bacteria using CAP and suggested a novel in-hospital disinfection alternative.

ObjectivesUsing a random forest algorithm, we previously found that teicoplanin-associated gene A (tcaA) might play a role in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactams, which we have investigated further here.MethodsRepresentative MRSA strains of prevalent clones were selected to identify the role of tcaA in the MRSA response to β-lactams. tcaA genes were deleted by homologous recombination in the selected MRSA strains, and antibiotic susceptibility tests were applied to evaluate the effect of tcaA on the minimum inhibitory concentrations (MICs) of glycopeptides and β-lactams. Scanning electron microscopy, RNA sequencing, and quantitative reverse transcription-polymerase chain reaction were performed to explore the mechanism of tcaA in MRSA resistance to β-lactams.ResultsThe MIC of penicillin plus clavulanate decreased from 3 mg/L to 0.064 mg/L and that of oxacillin decreased from 16 to 0.5 mg/L when tcaA was knocked out in the LAC strain. Compared with wild-type MRSA isolates, when tcaA was deleted, all selected strains were more susceptible to β-lactams. Susceptibility to ceftobiprole was restored in the ceftobiprole-resistant strain when tcaA was deleted. tcaA knockout caused “log-like” abnormal division of MRSA, and tcaA deficiency mediated low expression of mecA, ponA, and murA2.ConclusionsMachine learning is a reliable tool for identifying drug resistance-related genes. tcaA may be involved in S. aureus cell division and may affect mecA, ponA, and murA2 expression. Furthermore, tcaA is a potential resistance breaker target for β-lactams, including ceftobiprole, in MRSA.

Objectives: Fosfomycin has regained attention for treating infections caused by methicillin-resistant Staphylococcus aureus and multidrug-resistant coagulase-negative staphylococci. In this research, our objective was to investigate the mechanisms underlying fosfomycin resistance in Staphylococcus capitis . Methods: The minimum inhibitory concentrations (MICs) of fosfomycin were assessed in 109 clinical S. capitis isolates by the agar dilution method. By cloning the fos -like genes into the shuttle vector, pTSSCmPcap, and observing the change in fosfomycin MICs, the gene function was verified. Core genome multilocus sequence typing and comparative genomics analysis were conducted to determine the population characteristics of S. capitis isolates and analyse the genetic environment of the fos -like genes. Results: We identified a novel fosfomycin resistance gene, fosSC , on the chromosome in 58 out of 109 (53.2%) S. capitis isolates. The deduced products of the fosSC genes shared 67.15-67.88% amino acid sequence identity with FosB. The RN-pT- fos SC transformants carrying fosSC showed a 512-fold increase in the fosfomycin MICs. The fosSC gene was embedded in a conserved genetic context, but IS 431mec was located to the left of the fosSC gene in cluster L due to the insertion of staphylococcal cassette chromosome mec. Conclusions: The chromosomal fosSC genes in some lineages of S. capitis explained their high-level fosfomycin resistance. Ongoing surveillance is crucial for monitoring the potential threat of horizontal transfer, which could be facilitated by the presence of mobile genetic elements surrounding the fosSC gene. (c) 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

We read the article by Wendel et al. [1], which provides a novel mechanism underlying the emergence of daptomycin-resistant Staphylococcus aureus in clinical practice, with great interest. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide. Daptomycin (DAP) is the primary therapeutic choice for MRSA infections, and the emergence of DAP-resistant MRSA is a matter of substantial concern [2]. In a study performed by Wendel et al. [1], clinical DAP-resistant S. aureus isolates were confirmed, and DAP resistance was linked to antiseptic treatments.

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical Enterococcus faecium strain, SRR12, that carried a complete vanM gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical vanM gene cluster to that of SRR12 but a significantly increased vanM copy number in the genome. Long-read whole genome sequencing revealed that a one-copy vanM gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the vanM gene cluster jointed with IS1216E was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the vanM gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, vanM resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of vanM cluster amplification in the genome and an increased risk of vanM resistance dissemination.

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.

BackgroundSurgical sites infections (SSIs) caused by Methicillin-resistant Staphylococcus aureus (MRSA) constitute a major clinical problem. Understanding the transmission mode of MRSA is important for its prevention and control.AimWe investigated the transmission mode of a MRSA outbreak in a trauma and orthopedic hospital ward.MethodsClinical data were collected from patients (n = 9) with MRSA infection in a trauma and orthopedic ward from January 1, 2015 to December 31, 2019. The wards (n = 18), patients (n = 48), medical staff (n = 23), and their households (n = 5) were screened for MRSA. The transmission mode of MRSA isolates was investigated using next-generation sequencing and phylogenetic analyses. The resistance genes, plasmids, and single-nucleotide variants of the isolates were analyzed to evaluate microevolution of MRSA isolates causing SSIs. The MRSA colonization-positive doctor was asked to suspend his medical activities to stop MRSA spread.FindingsNine MRSA infected patients were investigated, of which three patients were diagnosed with SSI and had prolonged hospitalization due to the persistent MRSA infection. After screening, MRSA isolates were not detected in environmental samples. The surgeon in charge of the patients with SSI caused by MRSA and his son were positive for MRSA colonization. The MRSA from the son was closely related to the isolates detected in MRSA-induced SSIs patients with 8–9 single-nucleotide variants, while ST88-MRSA isolates with three different spa types were detected in the surgeon's nasal cavity. Comparative genomic analysis showed that ST88-MRSA isolates acquired mutations in genes related to cell wall synthesis, colonization, metabolism, and virulence during their transmission. Suspending the medical activity of this surgeon interrupted the spread of MRSA infection in this ward.ConclusionCommunity-associated MRSA clones can invade hospitals and cause severe postoperative nosocomial infections. Further MRSA surveillance in the households of health workers may prevent the transition of MRSA from colonization to infection.

ABSTRACT Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates ( n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC 50 = 1 µg/mL) than CC59 isolates (MIC 50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA ( P < 0.001). The agrA -I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR / walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Some of the most vexing problems of deep level relationship that remain in angiosperms involve the superrosids. The superrosid clade contains a quarter of all angiosperm species, with 18 orders in three subclades (Vitales, Saxifragales and core rosids) exhibiting remarkable morphological and ecological diversity. To help resolve deep-level relationships, we constructed a high-quality chromosome-level genome assembly for Tiarella polyphylla (Saxifragaceae) thus providing broader genomic representation of Saxifragales. Whole genome microsynteny analysis of superrosids showed that Saxifragales shared more synteny clusters with core rosids than Vitales, further supporting Saxifragales as more closely related with core rosids. To resolve the ordinal phylogeny of superrosids, we screened 122 single copy nuclear genes from genomes of 36 species, representing all 18 superrosid orders. Vitales were recovered as sister to all other superrosids (Saxifragales + core rosids). Our data suggest dramatic differences in relationships compared to earlier studies within core rosids. Fabids should be restricted to the nitrogen-fixing clade, while Picramniales, the Celastrales-Malpighiales (CM) clade, Huerteales, Oxalidales, Sapindales, Malvales and Brassicales formed an “expanded” malvid clade. The Celastrales-Oxalidales-Malpighiales (COM) clade (sensu APG IV) was not monophyletic. Crossosomatales, Geraniales, Myrtales and Zygophyllales did not belong to either of our well-supported malvids or fabids. There is strong discordance between nuclear and plastid phylogenetic hypotheses for superrosid relationships; we show that this is best explained by a combination of incomplete lineage sorting and ancient reticulation.

Background The overuse of antibiotics in livestock is contributing to the burden of antimicrobial resistance in humans, representing a One Health challenge. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has recently become a growing concern, and ST9 is the major LA-MRSA lineage in China and has emerged in clinical settings. Methods Antimicrobial susceptibility testing was used to evaluate the tetracycline resistance of ST9 MRSA collections, and gene cloning experiments were performed to explore the resistance mechanisms. Whole-genome sequencing and comparative genomics were used to analyse the genetic features of clinical ST9 isolates. A phylogenetic tree was constructed to investigate the relationship of human- and livestock-derived ST9 isolates. Results Clinical ST9 isolates were found to possess several types of resistance genes and resistance-related mutations and were multidrug-resistant. Notably, all clinical ST9 isolates were resistant to third-generation tetracyclines. Cloning experiments showed that both the acquisition of the tetracycline resistance gene tet(L)/tet(63) and a mutation in the rpsJ gene contributed to third-generation tetracycline resistance. Phylogenetic analysis showed that the ST9 isolates collected in healthcare systems were probably transmitted from livestock. The ST9 lineage underwent multiple interspecies recombination events and gained many resistance elements. Furthermore, the resistance to third-generation tetracyclines may have evolved under tetracycline pressure in livestock. Conclusions The evolution of ST9 MRSA in livestock and transmission of this clone between humans and livestock highlight the importance of establishing control strategies with the One Health approach to reduce the burden of antibiotic resistance.

ObjectiveTo determine the genetic structure of ermB-positive Tn1546-like mobile elements in methicillin-resistant Staphylococcus aureus (MRSA) from mainland China. MethodsA total of 271 erythromycin-resistant MRSA isolates were isolated from Sir Run Run Shaw Hospital (SRRSH) from 2013 to 2015. Whole-genome sequencing was performed for the ermB-positive strains, and the genetic environment of the ermB genes was analyzed. Southern hybridization analysis and transformation tests were performed to confirm the location of the ermB gene. ResultsA total of 64 isolates (64/271, 23.6%) were ermB-positive strains, with 62 strains (62/64, 96.9%) belonging to the CC59 clone. The other two strains, SR130 and SR231, belonging to CC5-ST965, both harbored 14,567 bp ermB-positive Tn1546-like elements and displayed multidrug-resistant profiles. PFGE followed by Southern blot demonstrated that the ermB genes were located on the plasmids of both SR130 and SR231, while two copies of ermB were located on the chromosome of SR231. Further sequencing demonstrated that SR231 carried one Tn1546-ermB elements in the plasmid and two identical copies integrated on the chromosome, which had 99.99% identity to the element in the plasmid of SR130. The Tn1546-ermB elements were highly similar (100% coverage, >99.9% identity) to the element Tn6636 reported in a previous study from Taiwan. The plasmids (pSR130 and pSR231) harboring ermB-positive Tn1546-like elements were also identical to the mosaic plasmid pNTUH_5066148. However, conjugation of ermB-carrying plasmids of SR130 and SR231 were failed after triple repeats. ConclusionMultiple copies of ermB-positive Tn1546-like mobile elements were found in CC5-ST965 MRSA from mainland China, showing the wide dissemination of these Enterococcus faecium-originated ermB-positive Tn1546-like elements. Molecular epidemiological study of Tn1546-like elements is essential to avoid the spreading of resistant determinants.

Fosfomycin has gained attention as a combination therapy for methicillin-resistant Staphylococcus aureus infections. Hence, the detection of novel fosfomycin-resistance mechanisms in S. aureus is important. Here, the minimal inhibitory concentrations (MICs) of fosfomycin in CC1 methicillin-resistant S. aureus were determined. The pangenome analysis and comparative genomics were used to analyse CC1 MRSA. The gene function was confirmed by cloning the gene into pTXΔ. A phylogenetic tree was constructed to determine the clustering of the CC1 strains of S. aureus. We identified a novel gene, designated fosY, that confers fosfomycin resistance in S. aureus. The FosY protein is a putative bacillithiol transferase enzyme sharing 65.9-77.5% amino acid identity with FosB and FosD, respectively. The function of fosY in decreasing fosfomycin susceptibility was confirmed by cloning it into pTXΔ. The pTX-fosY transformant exhibited a 16-fold increase in fosfomycin MIC. The bioinformatic analysis showed that fosY is in a novel genomic island designated RIfosY (for "resistance island carrying fosY") that originated from other species. The global phylogenetic tree of ST1 MRSA displayed this fosY-positive ST1 clone, originating from different regions, in the same clade. The novel resistance gene in the fos family, fosY, and a genomic island, RIfosY, can promote cross-species gene transfer and confer resistance to CC1 MRSA causing the failure of clinical treatment. This emphasises the importance of genetic surveillance of resistance genes among MRSA isolates.

Staphylococcus capitis , which causes bloodstream infections in neonatal intensive care units, is a common cause of healthcare-associated infections. Thus, a standardized high-resolution typing method to document the transmission and dissemination of multidrug-resistant S. capitis isolates is required. We aimed to establish a core genome multilocus sequence typing (cgMLST) scheme to surveil S. capitis . The cgMLST scheme was defined based on primary and validation genome sets and tested with outbreaks of linezolid-resistant isolates and a validation set. Phylogenetic analysis was performed to investigate the population structure and compare it with the result of cgMLST analysis. The S. capitis population consists of 1 dominant, NRCS-A, and 4 less common clones. In this work, a multidrug-resistant clone (L clone) with linezolid resistance is identified. With the features of type III SCC mec and multiple copies of mutations of G2576T and C2104T in the 23S rRNA, the L clone has been spreading silently across China.