Role of tcaA, a potential target as a ceftobiprole resistance breaker, in MRSA ß-lactam resistance.

OBJECTIVES: Using 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 ß-lactams, which we have investigated further here. METHODS: Representative MRSA strains of prevalent clones were selected to identify the role of tcaA in the MRSA response to ß-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 ß-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 ß-lactams. RESULTS: The 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 ß-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. CONCLUSIONS: Machine 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 ß-lactams, including ceftobiprole, in MRSA.

Identification of the novel fosfomycin resistance gene fosSC in Staphylococcus capitis.

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, pTSSCm-Pcap, 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-fosSC transformants carrying fosSC showed a 512-fold increase in the fosfomycin MICs. The fosSC gene was embedded in a conserved genetic context, but IS431mec 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.

Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae.

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum ß-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.IMPORTANCEThis study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel ß-lactam/ß-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

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 (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 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.

Exploring the third-generation tetracycline resistance of multidrug-resistant livestock-associated methicillin-resistant Staphylococcus aureus ST9 across healthcare settings in China.

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.

Microstructural Characterization of the As-Cast and Homogenized Al-Cu-Mg-Ag Alloy.

In this study, the as-cast microstructure and the evolution of the homogenized microstructure of large-scale industrialized Al-Cu-Mg-Ag heat-resistant aluminum alloy ingots were investigated by means of optical microscopy (OM), scanning electron microscopy (SEM), energy dispersive analysis (EDS), and differential scanning calorimetry (DSC). The results indicate that the dendritic segregation is evident in the ingot along the radial direction, and the grain boundaries are decorated with lots of net-shaped continuous eutectic structures. With the homogenization time extension and the homogenization temperature increase, the eutectic phases (i.e., the primary Al2Cu phase, the Al2CuMg phase, and the AlCuMgAg quaternary phase) at the grain boundaries gradually dissolve back into the matrix. Meanwhile, most of the dendritic grain boundaries gradually become sparse and thinner. Finally, it is found that the optimal homogenization regime of the Al-Cu-Mg-Ag alloy is 420 °C/5 h+480 °C/8 h+515 °C/24 h.

Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1.

OBJECTIVES: This study aims to investigate the effect of morinda officinalis polysaccharides (MOP) in inflammatory microenvironment on the expression of silent information regulator sirtuin 1 (SIRT1) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in periodontal ligament cells. METHODS: Thirty rats were randomly divided into control group (n=6) and model group (n=24). The model group used orthodontic wire ligation to establish periodontitis, and six rats from each group were killed after 3 weeks. The successful modeling was confirmed by Micro-CT. The remaining rats in the model group were randomly divided into natural recovery group, normal saline (NS) group, and MOP group. In the MOP group, MOP [200 mg/(kg·3d), 50 µL for 4 weeks] was injected into the palatal side of the left maxillary first molar of the rats, while the NS group was injected with equal volume of NS. The natural recovery group did not undergo any treatment. The left maxilla tissues of the rats were collected, and pathological changes in perio-dontal ligament cells were observed by hematoxylin-eosin (HE) staining. The expression of SIRT1 and NLRP3 was detected by immunohistochemistry. Cultivate periodontal ligament fibroblasts in vitro and detect the effect of MOP on cell activity using CCK-8. The 4th generation cells were divided into control group, inflammation group (10 µg/mL lipopolysaccharide), and experimental group (5 µmol/L MOP, 5 µmol/L MOP+10 µg/mL lipopolysaccharide). The expression of SIRT1 and NLRP3 was detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot analyses. The acetylation of NLRP3 and the contents of interleukin (IL)-1ß and IL-18 were detected by immunoprecipitation and enzyme-linked immunosorbent assay, respectively. Statistical analysis of data was conducted using Prism 9.0 software. RESULTS: In the vivo experiments, the expression of NLRP3 and SIRT1 in the MOP group decreased significantly compared with that in the natural recovery group and NS group, while the expression of SIRT1 increased (P<0.05) and inflammatory cell infiltration decreased. In the in vitro experiments, the expression of NLRP3 mRNA and protein in the inflammation group increased (P<0.05), while the expression of SIRT1 significantly decreased (P<0.01); MOP upregulated the expression of SIRT1 in inflammatory cells (P<0.05), reduced the expression of NLRP3 and its acetylation level significantly (P<0.05), suppressed the content of IL-1ß and IL-18 in the supernatant (P<0.01). CONCLUSIONS: The SIRT1 expression decreased, and that of NLRP3 expression increased in inflammatory periodontal ligament cells. MOP intervention promoted SIRT1 expression, resulting in the inhibition of NLRP3. Meanwhile, the acetylation level of NLRP3 reduced through deacetylation, leading to the decreased activity of NLRP3. Thus, MOP acted as inflammatory suppressor.

Antifungal susceptibility and molecular characteristics of Cryptococcus spp. based on whole-genome sequencing in Zhejiang Province, China.

Cryptococcus spp. is a complex species that often causes cryptococcosis, which is one of the most common opportunistic infections in adults living with HIV and has very high morbidity and mortality rates. This study aimed to investigate the antifungal susceptibility profiles and epidemiological characteristics of the Cryptococcus neoformans species complex (CNSC) and the Cryptococcus gattii species complex (CGSC) in Zhejiang Province, China. A total of 177 CNSC and 3 CGSC isolates were collected, and antifungal susceptibility was tested by FUNGUS 3 and verified with an E-test. Moreover, multiple classification methods and genomic analyses were performed. The majority of the isolates (96.11%) were C. neoformans (formerly C. neoformans var. grubii) (ST5-VNI-A-α). Our study highlights that most of the patients with cryptococcosis were non-HIV patients in China, and nearly half of them did not have underlying diseases that led to immune insufficiency. Most of the Cryptococcus spp. isolates in this study were sensitive to common antifungal drugs. Two 5-flucytosine (5-FC)-resistant strains were identified, and FUR1 mutation was detected in the 5-FC-resistant isolates. Typing based on whole-genome sequencing (WGS) showed better discrimination than that achieved with multilocus sequence typing (MLST) and indicated a clear population structure. A phylogenetic analysis based on WGS included more genomic information than traditional classification methods.

The Genetic Characteristics and Carbapenem Resistance Mechanism of ST307 Klebsiella pneumoniae Coharbouring blaCMY-6, blaOXA-48, and a Truncated blaNDM-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a common nosocomial pathogen causing severe infectious diseases, and ST307 CRKP is an emerging clone. In this study, we collected five ST307 CRKP isolates, evaluated their antimicrobial susceptibility using microbroth dilution, and their clonality and population structure by PFGE, cgMLST, and SNP-based phylogenetic analysis. Then, the genome characteristics, such as antimicrobial resistance genes and plasmid profiles, were studied by subsequent genomic analysis. The plasmid transfer ability was evaluated by conjugation, and the carbapenem resistance mechanism was elucidated by gene cloning. The results showed that all five ST307 CRKP isolates harboured blaCMY-6, blaOXA-48, and blaNDM-1; however, the end of the blaNDM-1 signal peptide was interrupted and truncated by an IS10 element, resulting in the deactivation of carbapenemase. The ST307 isolates were closely related, and belonged to the globally disseminated clade. blaOXA-48 and blaNDM-1 were located on the different mobilisable IncL/M- and IncA/C2-type plasmids, respectively, and either the pOXA-48 or pNDM-1 transconjugants were ertapenem resistant. Gene cloning showed that blaCMY-6 could elevate the MICs of carbapenems up to 64-fold and was located on the same plasmid as blaNDM-1. In summary, ST307 is a high-risk clone type, and its prevalence should be given additional attention.

Development of a novel core genome MLST scheme for tracing multidrug resistant Staphylococcus capitis.

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 SCCmec and multiple copies of mutations of G2576T and C2104T in the 23S rRNA, the L clone has been spreading silently across China.

Staphylococcal cassette chromosome mec amplification as a mechanism for ceftobiprole resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

OBJECTIVES: In this study, we evaluated the ceftobiprole (BPR) susceptibilities of 472 methicillin-resistant Staphylococcus aureus (MRSA) isolates, and investigated the mechanisms underlying BPR resistance. METHODS: For all MRSA isolates, BPR MIC was determined by agar dilution. We sequenced the BPR-resistant isolates through Illumina short- and MinION long-read sequencing. We also selected MRSA isolates of ST5, ST59, and ST239, and exposed them to increasing BRP concentrations. The isolated mutants developing BPR resistance were sequenced. RESULTS: A total of 471 MRSA isolates were susceptible to BPR, with MICs ranging from 0.25 to 2 mg/L. Compared with HA-MRSA isolates (MIC50 = 2 mg/L; MIC90 = 2 mg/L), CA-MRSA isolates (MIC50 = 0.5; MIC90 = 2 mg/L) were more susceptible to BPR (p < 0.001). Compared with isolates with staphylococcal cassette chromosome mec (SCCmec) type II or III (MIC50 = 2 mg/L; MIC90 = 2 mg/L), isolates with SCCmec type IV (MIC50 = 1 mg/L; MIC90 = 1 mg/L) or V (MIC50 = 0.5 mg/L; MIC90 = 1 mg/L) were more susceptible to BPR (p < 0.001). Nanopore sequencing revealed two copies of SCCmec repeats in the BPR-resistant MRSA isolate. In addition, SCCmec amplification could be induced by BPR exposure in ST239 MRSA isolates; however, no amplification was observed in the other lineages. The induced BPR-resistant MRSA isolates also acquired mutations in mecA and other genes, such as guaA, guaB, relA, rpoA, and oatA, which were speculated as factors contributing to BPR-resistance development. DISCUSSION: BPR showed significant antibacterial activity against MRSA isolates in China; however, the emergence of a BPR-resistant isolate before its launch was a cause for concern. Multiple genes and pathways are potentially involved in the development of BPR resistance in MRSA, and our data demonstrated the role of nanopore-sequencing in revealing the tandem repeat-mediated resistance mechanism in MRSA.

The novel fosfomycin resistance gene fosY is present on a genomic island in CC1 methicillin-resistant Staphylococcus aureus.

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.

Genetic Characteristics of Multiple Copies of Tn1546-Like Elements in ermB-Positive Methicillin-Resistant Staphylococcus aureus From Mainland China.

Objective: To determine the genetic structure of ermB-positive Tn1546-like mobile elements in methicillin-resistant Staphylococcus aureus (MRSA) from mainland China. Methods: A 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. Results: A 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. Conclusion: Multiple 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.

Epidemiology, evolution and cryptic susceptibility of methicillin-resistant Staphylococcus aureus in China: a whole-genome-based survey.

OBJECTIVES: The aim of this study was to investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in China to identify predominant lineages and their associations with clinical data and antimicrobial resistance profiles. METHODS: We performed a national prevalence study of patients with S. aureus infections in 22 tertiary hospitals in China from 2015 to 2017. Clinical data from patients and the antimicrobial phenotypes were collected for each isolate. Genome sequencing was performed on a proportion of isolates and a phylogenetic analysis was undertaken. Genotypic and phenotypic ß-lactam susceptibilities were compared. RESULTS: A total of 1900 patients with S. aureus infections were included, of which 40% involved MRSA. Community-associated MRSA (CA-MRSA) infections were 24% of the total isolates. Genomic data showed that more than three-quarters of the MRSA were from three dominant lineages CC239 (25%, 116/471), CC5 (21%, 96/471) and CC59 (33%, 154/471) with CC59 accounting for more than half of the CA-MRSA isolates. Penicillin susceptibility genomic features were observed in 53% (251/470) of MRSA, including almost all of the CC59 (152/154) lineage, and 96% (242/251) of these isolates demonstrated in vitro susceptibility to penicillin or amoxicillin combined with clavulanic acid. Phylogenetic analysis indicated that the CC59 lineage can be divided into six lineages with all Asian CC59 isolates likely arising from an ancestral Mainland China lineage. CONCLUSIONS: This study showed a high prevalence of CA-MRSA in China, largely due to the widespread presence of CC59. As almost all isolates in this lineage possess genetic variants leading to increased ß-lactam susceptibility, we suggest that to improve antibiotic stewardship combinations of penicillins and ß-lactamase inhibitors should be included in the antibiotic susceptibility testing panels used to inform treatment decisions and research undertaken on this combination therapy.

Transmission and microevolution of methicillin-resistant Staphylococcus aureus ST88 strain among patients, healthcare workers, and household contacts at a trauma and orthopedic ward.

Background: Surgical 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. Aim: We investigated the transmission mode of a MRSA outbreak in a trauma and orthopedic hospital ward. Methods: Clinical 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. Findings: Nine 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. Conclusion: Community-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.

A random forest model based on core genome allelic profiles of MRSA for penicillin plus potassium clavulanate susceptibility prediction.

Treatment failure of methicillin-resistant Staphylococcus aureus (MRSA) infections remains problematic in clinical practice because therapeutic options are limited. Penicillin plus potassium clavulanate combination (PENC) was shown to have potential for treating some MRSA infections. We investigated the susceptibility of MRSA isolates and constructed a drug susceptibility prediction model for the phenotype of the PENC. We determined the minimum inhibitory concentration of PENC for MRSA (n=284) in a teaching hospital (SRRSH-MRSA). PENC susceptibility genotypes were analysed using a published genotyping scheme based on the mecA sequence. mecA expression in MRSA isolates was analysed by qPCR. We established a random forest model for predicting PENC-susceptible phenotypes using core genome allelic profiles from cgMLST analysis. We identified S2-R isolates with susceptible mecA genotypes but PENC-resistant phenotypes; these isolates expressed mecA at higher levels than did S2 MRSA (2.61 vs 0.98, P<0.05), indicating the limitation of using a single factor for predicting drug susceptibility. Using the data of selected UK-sourced MRSA (n=74) and MRSA collected in a previous national survey (NA-MRSA, n=471) as a training set, we built a model with accuracies of 0.94 and 0.93 for SRRSH-MRSA and UK-sourced MRSA (n=287, NAM-MRSA) validation sets. The AUROC of this model for SRRSH-MRSA and NAM-MRSA was 0.96 and 0.97. Although the source of the training set data affects the scope of application of the prediction model, our data demonstrated the power of the machine learning approach in predicting susceptibility from cgMLST results.

Household Transmission of Community-Associated Methicillin-Resistant Staphylococcus Aureus.

Currently, the mechanism of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) transmission mechanism is unclear; however, it must be considered in conjunction with asymptomatic S. aureus strains colonization dynamics. This epidemiological study aimed to determine the role of the household in CA-MRSA transmission in China. Five patients with culture-confirmed CA-MRSA infection and five control patients were recruited from the Sir Run Run Shaw Hospital in Zhejiang, China, between December 2019 and January 2020. The household members of the patients, their pets, and environmental surfaces were sampled and screened for MRSA colonization. Mass spectrometry identification and antimicrobial susceptibility testing were performed on the MRSA isolates. Whole-genome sequencing and core genome multilocus sequence typing (cgMLST) were performed to determine the origin and transmission of the MRSA isolates in the households. Overall, 14 S. aureus-positive specimens (14.1%, 14/99) were obtained from the five households of patients with CA-MRSA infections, of which 12 (85.7%) were MRSA. The overall positivity of MRSA was 12.1% (12/99) among the samples from the CA-MRSA households, while no MRSA isolates were detected in the five control households. Most MRSA isolates belonged to epidemic CA-MRSA clones, such as ST59 (15/35, 42.9%) and ST508 (15/35, 42.9%). The cgMLST results confirmed that MRSA was transmitted among patients, contacts, and pets in the households and was present on environmental surfaces in the CA-MRSA patients' households. In conclusion, the study revealed that the home environment was an important MRSA reservoir. Therefore, focusing on MRSA decolonization in patients alone is not sufficient for infection control of CA-MRSA.

[Different interbody fustion cages and combined fixation through intermuscular approach for lumbar diseases:a case control study].

OBJECTIVE: To explore the clinical effect of combined fixation and interbody bone grafting through intermuscular approach with different fusion cages in the treatment of single segment lumbar diseases. METHODS: From June 2014 to December 2016, the clinical data of 123 cases of single segment lumbar diseases were analyzed retrospectively, including 44 males and 79 females, aged 22 to 60 years old, with the diseases course of 6 to 84 months. The disease types involved lumbar disc degeneration in 65 cases, lumbar spinal stenosis in 30 cases, MeyerdingⅠslip in 21 cases, giant lumbar disc herniation in 7 cases. Lesions was L3, 4 in 5 cases, L4, 5 in 101 cases, L5S1 in 17 cases. According to the application of different interbody fusion cage, patients were divided into single common cage group, double common cage group and banana type cage group. The operation time, intraoperative hemorrhage, postoperative incision drainage fluid and incision length were observed in three groups; VAS score of lumbar incision and JOA score of preoperative and final follow up were recorded 72 hours after operation;the intervertebral space height, Cobb angle of lumbar coronal and sagittal plane before and after operation, and interbody fusion 12 months after operation were observed by imaging data. RESULTS: There was no significant difference in incision length, operation time, intraoperative bleeding volume, postoperative drainage volume and VAS score of lumbar incision 72 hours after operation among three groups (P>0.05). All cases were followed up for 12 to 36 (23.70±4.52) months. The height of intervertebral space in three groups recovered significantly (P<0.05), at the last follow-up, there were different degrees of loss, the loss degree of single common fusion cage group>banana type fusion cage group>double common fusion cage group. At the last follow-up, the Cobb angle in the coronaland sagittal planes of the three groups were significantly improved (P<0.05). During the follow-up, there were 42 cases of fusion cage subsidence, including 26 cases in the single common cage group, 5 cases in the double common cage group, 11 cases in the banana cage group, the difference was statistically significant (P<0.05). At 12 months after operation, the interbody fusion rate was 83% in the single common cage group, 95% in the double common cage group and 90% in the banana cage group, the interbody fusion rate in the two common cage group and the banana cage group was better than that in the single common cage group. No obvious degeneration was observed in the adjacent segments. At the last follow-up, the JOA scores of the three groups were statistically significant (P<0.05). The incidence of single common fusion cage group was 10%(4 / 42), that of double common fusion cage group was 9%(4 / 43), and that of banana fusion cage group was 10%(4 / 39). There was no significant difference among the three groups. CONCLUSION: Through the intermuscular approach, single pedicle screw and contralateral facet screw were used for fixation, and single common fusion cage, double common fusion cage or banana type fusion cage were used for interbody grafting to treat single segment lumbar disease. Although the application of different fusion cage could not increase the axial strength of fixed segment, the speed of fusion was accelerated by increasing the contact area, and the quality of the fusion cage reduces the settlement of the cage and the loss of the height of the intervertebral space. Therefore, two interbody fusion cages implanted in one side are of positive clinical significance for the fixation of unilateral pedicle screw combined with contralateral facet screw, without prolonging the operation time and increasing the complications.

Predominance of ST5-II-t311 clone among healthcare-associated methicillin-resistant Staphylococcus aureus isolates recovered from Zhejiang, China.

OBJECTIVES: To determine the molecular characteristics and antimicrobial susceptibility of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in Zhejiang Province. METHODS: A total of 391 HA-MRSA isolates were collected from 12 hospitals in five cities of Zhejiang Province, between January 2012 and May 2013. Susceptibility to vancomycin, teicoplanin, linezolid, tigecycline, and daptomycin was determined. Resistant isolates were screened for resistance mutations. Ten isolates from each hospital were then chosen at random for molecular typing. RESULTS: The isolates showed good susceptibility to all five anti-MRSA agents; only five sporadic non-susceptible isolates were detected. CC5/ST5-MRSA-II-t311 (39/120, 32.5%) was found to be the predominant HA-MRSA clone and was spread between the different hospitals in Hangzhou. CC5/ST5-MRSA-II-t002 was the most prevalent clone in Ningbo, while CC239/ST239-MRSA was epidemic only in certain hospitals in Wenzhou and Shaoxing. Fifteen ST59 isolates (15/120, 12.5%) were identified among the HA-MRSA isolates. CONCLUSIONS: CC5/ST5-MRSA-II-t311 has become the predominant HA-MRSA clone in Hangzhou, Zhejiang Province. ST59 MRSA has spread into hospitals. The isolates showed good susceptibility to all five anti-MRSA agents.

Survival and regeneration of deep-freeze preserved autologous cranial bones after cranioplasty.

BACKGROUND: After decompressive craniectomy, a deep-freeze-preserved autologous cranial bone graft can be used for cranioplasty to avoid immunoreaction against an artificial patch material. Autologous cranial bone grafts not only have better physical properties, such as heat conduction, compared to artificial patch materials, but they also have the advantages of a lower medical cost and satisfactory physical flexibility. The discussion over (99)Tc(m)-MDP SPECT static cranial bone tomography in the diagnosis of survival and regeneration in deep-freeze preservation autologous cranial bones after cranioplasty is valuable. Objective. To investigate whether deep-freeze-preserved autologous cranial bone grafts could survive and regenerate after autologous reimplantation. METHODS: The method of cranial bone preservation involved removing the cranial graft and sealing it in a double-layer sterile plastic bag under sterile surgical conditions. On the day of the cranioplasty operation, the cranial bone graft was disinfected by immersing it in 3% povidone-iodine for 30 minutes. At short-term (2 weeks), medium-term (3 months), and long-term (12 months) postoperative follow-up visits, (99)Tc(m)-MDP SPECT static cranial bone tomography was used to examine the reimplanted cranial bone. Results. There were no postoperative infections or seromas in all 16 cases. Two weeks following cranial bone graft reimplantation, the SPECT tomography showed some radioactivity uptake in the reimplanted cranial bone graft, which was lower than that in the cranial bone on the healthy side. At 3 months and 12 months after the operation, the radioactivity uptake in the reimplanted cranial bone graft was the same as that in the cranial bone on the healthy side. X-ray films showed blurred sutures in the reimplanted cranial bone graft at 12 months after surgery. CONCLUSION: Reimplanted deep-freeze-preserved autologous cranial bone can survive in the short term and regenerate in the medium and long terms.