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Abstract Background In recent years, the hypervirulent and carbapenem-resistant Klebsiella pneumoniae has been increasingly reported worldwide. The objective of this study was to compare the antibiotic resistance and virulence profiles of carbapenem-resistant hypervirulent K.pneumoniae (CR-hvKP) and hypervirulent carbapenem-resistant K.pneumoniae (hv-CRKP) and identify the prevailing strain in clinical settings. Methods In this study, hv-CRKP or CR-hvKP were identified based on the results of whole-genome analysis (WGS), multilocus sequence typing (MLST) and the antimicrobial susceptibility testing. We then compared antibiotic resistance and virulence profiles between CR-hvKP and hv-CRKP through the antimicrobial susceptibility testing and a series of virulence experiments including biofilm formation ability detection method, the resistance test against human serum, siderophore production test, neutrophil phagocytosis assay and Galleria mellonella infection model. Additionally, pathway enrichment analysis was conducted to assess the effect of SNPs on the phenotype. Results In this study, we categorized 17.4% of hypervirulent and carbapenem-resistant K. pneumoniae strains as CR-hvKP and 82.6% as hv-CRKP. Among them, 84.2% (16/19) of CR-hvKP strains harboring carbapenemase genes exhibited lower imipenem and meropenem MIC values compared to hv-CRKP strains. The virulence potential of hv-CRKP and CR-hvKP was confirmed by using virulence experiments in vitro and in vivo, showing that virulence of the CR-hvKP strains was comparable to that of hv-CRKP strains. Notably, the 90 hv-CRKP strains were classified into 3 different ST types and 8 capsule types, each showing varying degrees of resistance and virulence. We observed that subclonal replacement was within the predominant hv-CRKP clone, with the ST11-KL64 strain, characterized by high-level resistance and virulence emerging as the currently prevailing subclone, replacing ST11-KL47. KEGG enrichment analysis showed that pathways associated with the citrate cycle (TCA cycle), glycolysis/gluconeogenesis, glutathione metabolism, two-component regulatory system, and folate metabolism were significantly enriched among the group expressing different levels of capsular polysaccharides. Conclusions The hv-CRKP strains exhibited a greater survival advantage in the hospital environment than CR-hvKP strains. Notably, the ST11-KL64 hv-CRKP strain which displayed a high level of resistance and hypervirulence, warrants the most clinical vigilance. Clinical trial number Not applicable.

Abstract Background Staphylococcus aureus, a commensal bacterium, colonizes the skin and mucous membranes of approximately 30% of the human population. Apart from conventional resistance mechanisms, one of the pathogenic features of S. aureus is its ability to survive in a biofilm state on both biotic and abiotic surfaces. Due to this characteristic, S. aureus is a major cause of human infections, with Methicillin-Resistant Staphylococcus aureus (MRSA) being a significant contributor to both community-acquired and hospital-acquired infections. Results Analyzing non-repetitive clinical isolates of MRSA collected from seven provinces and cities in China between 2014 and 2020, it was observed that 53.2% of the MRSA isolates exhibited varying degrees of ability to produce biofilm. The biofilm positivity rate was notably high in MRSA isolates from Guangdong, Jiangxi, and Hubei. The predominant MRSA strains collected in this study were of sequence types ST59, ST5, and ST239, with the biofilm-producing capability mainly distributed among moderate and weak biofilm producers within these ST types. Notably, certain sequence types, such as ST88, exhibited a high prevalence of strong biofilm-producing strains. The study found that SCCmec IV was the predominant type among biofilm-positive MRSA, followed by SCCmec II. Comparing strains with weak and strong biofilm production capabilities, the positive rates of the sdrD and sdrE were higher in strong biofilm producers. The genetic determinants ebp, icaA, icaB, icaC, icaD, icaR, and sdrE were associated with strong biofilm production in MRSA. Additionally, biofilm-negative MRSA isolates showed higher sensitivity rates to cefalotin (94.8%), daptomycin (94.5%), mupirocin (86.5%), teicoplanin (94.5%), fusidic acid (81.0%), and dalbavancin (94.5%) compared to biofilm-positive MRSA isolates. The biofilm positivity rate was consistently above 50% in all collected specimen types. Conclusions MRSA strains with biofilm production capability warrant increased vigilance.

The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced hemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and hemolytic activity in S. aureus.

Fluoroquinolones (FQ) are essential for the treatment of multidrug-resistant tuberculosis (MDR-TB). The FQ resistance (FQ-R) rate in MDR-TB in China and its risk factors remain poorly understood. We conducted a retrospective, population-based genomic epidemiology study of MDR-TB patients in Shanghai, China, from 2009 to 2018. A genomic cluster was defined as strains with genetic distances ≤ 12 single nucleotide polymorphisms. The transmitted FQ-R was defined as the same FQ resistance-conferring mutations shared by ≥ 2 strains in a genomic cluster. We used multivariable logistic regression analysis to identify the risk factors for drug resistance. Among the total 850 MDR-TB patients included in the study, 72.8% (619/850) were male, the median age was 39 (interquartile range 28, 55) years, 52.7% (448/850) were migrants, and 34.5% (293/850) were previously treated patients. Most of the MDR-TB strains belong to the Beijing lineage (91.7%, 779/850). Overall, the genotypic resistance rate of FQ was 34.7% (295/850), and 47.1% (139/295) FQ-R patients were in genomic clusters, of which 98 (33.2%, 98/295) were presumed as transmitted FQ-R. Patients with treatment-naïve (aOR = 1.84; 95% CI: 1.09, 3.16), diagnosed in a district-level hospital (aOR = 2.69; 95% CI: 1.56, 4.75), and streptomycin resistance (aOR = 3.69; 95% CI: 1.65, 9.42) were significantly associated with the transmission of FQ-R. In summary, the prevalence of FQ-R among MDR-TB patients was high in Shanghai, and at least one-third were transmitted. Enforced interventions including surveillance of FQ drug susceptibility testing and screening among MDR-TB before initiation of treatment were urgently needed.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that could cause life-threatening bloodstream infections. The objective of this study was to identify potential diagnostic biomarkers of S. aureus bloodstream infection. Gene expression dataset GSE33341 was optimized as the discovery dataset, which contained samples from human and mice. GSE65088 dataset was utilized as a validation dataset. First, after overlapping the differentially expressed genes (DEGs) in S. aureus infection samples from GSE33341-human and GSE33341-mice samples, we detected 63 overlapping genes. Subsequently, the hub genes including DRAM1, PSTPIP2, and UPP1 were identified via three machine-learning algorithms: random forest, support vector machine-recursive feature elimination, and least absolute shrinkage and selection operator. Additionally, the receiver operating characteristic curve was leveraged to verify the efficacy of the hub genes. DRAM1 (AUC=1), PSTPIP2 (AUC=1), and UPP1 (AUC=1) were investigated and demonstrated significant expression differences (all P < 0.05) and diagnostic efficacy in the training and validation datasets. Furthermore, the relationship between the diagnostic markers and the abundance of immune cells was assessed using cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). These three diagnostic indicators also correlated with multiple immune cells to varying degrees. The expression of DRAM1 was significantly positively correlated with B cell naive and mast cell activation, and negatively correlated with NK cells and CD4/CD8+ T cells. The expression of PSTPIP2 was significantly positively correlated with macrophage M0, macrophage M1, B cell naive, and dendritic cell activation, while the expression of PSTPIP2 was negatively correlated with NK cells and CD4/CD8+ T cells. Significant negative correlations between UPP1 expression and T cell CD4 memory rest and neutrophils were also observed. Finally, we established a mouse model of S. aureus bloodstream infection and collected the blood samples for RNA-Seq analysis and RT-qPCR experiments. The analysis results in RNA-Seq and RT-qPCR experiments further confirmed the significant expression differences (all P < 0.05) of these three genes. Overall, three candidate hub genes (DRAM1, PSTPIP2, and UPP1) were identified initially for S. aureus bloodstream infection diagnosis. Our study could provide potential diagnostic biomarkers for S. aureus bloodstream infection patients.

ABSTRACT The widespread prevalence and dissemination of antibiotic-resistant bacteria, coupled with the diminishing supply of new antibiotics, emphasize the pressing necessity for the exploration of innovative antibacterial agents. Previously, we detailed the impact of the small-molecule compound CY-158-11 on S. aureus biofilm. By hindering adhesion and PIA-mediated biofilm formation, subinhibitory concentrations of CY-158-11 exhibit antibiofilm activity toward S. aureus. Here, we sought to elucidate the antibacterial activity and mode of action of this compound. Upon CY-158-11 treatment in culture, the inhibition of bacterial growth, coupled with MBC to MIC of >4, indicated that CY-158-11 exerted a bacteriostatic effect. Particularly, CY-158-11 showed strong antibacterial activity against a wide variety of S. aureus, including multidrug-resistant bacteria. We found that CY-158-11 promoted the permeability of cell membrane and propidium iodide absorption as well as caused the dissipation of membrane potential. The effect of CY-158-11 on the mammalian cytoplasmic membrane was measured using hemolytic and cytotoxicity assays, and the skin irritation and systemic toxicity of the drug were measured by injecting the compound into the skin and tail vein of mice. Moreover, CY-158-11 exhibited considerable efficacy in a subcutaneous abscess mouse model of S. aureus infection. In conclusion, CY-158-11 possesses antibacterial properties, including inhibition of bacterial growth, damage to cell membranes, and treatment of skin abscesses, which can be a promising therapeutic option for combating S. aureus.IMPORTANCEThe combination of the rising incidence of antibiotic resistance and the shrinking antibiotic pipeline has raised concern about the postantibiotic era. New antibacterial agents and targets are required to combat S. aureus-associated infections. In this study, we identified a maleimide-diselenide hybrid compound CY-158-11 exhibiting antibacterial activity against S. aureus in vitro and in vivo at relatively low concentrations. Furthermore, the investigation of its mode of action revealed that CY-158-11 can selectively perturb the cytoplasmic membrane of bacteria without harming mammalian cells or mouse organs. Thus, CY-158-11 is a compelling novel drug for development as a new therapy for S. aureus infections.

BackgroundDaptomycin non-susceptible Staphylococcus aureus (DNS) strains pose a serious clinical threat, yet their characteristics remain poorly understood.MethodsDNS derivatives were generated by exposing S. aureus strains to subinhibitory concentrations of daptomycin. Competition experiment and growth kinetics experiment were used to observe the growth of bacteria. Galleria mellonella larvae and mouse skin abscess models were used to observe the virulence of bacteria. Transmission electron microscopy (TEM), cytochrome C experiment and biofilm formation experiment were used to observe the drug resistance phenotype. And homologous recombination was used to study the role of mutations.ResultsPhenotypic profiling of DNS strains revealed impaired growth, increased cell wall thickness, enhanced biofilm formation, reduced negative surface charge, and attenuated virulence compared to their wild-type strains. Whole genome sequencing identified mutations in mprF, cls2, and saeR in DNS strains. Allelic replacement experiments validated the roles of MprF L341F and Cls2 F60S substitutions in augmenting daptomycin non-susceptibility in Newman. Deletion of saeR in the NewmanMprFL341F strain and complementation of saeR in the Newman-DNS strain did not directly alter daptomycin susceptibility. However, the deletion of saeR was found to enhance competitive fitness under daptomycin pressure.ConclusionThis work validates adaptive laboratory evolution (ALE) for modeling clinical DNS strains and uncovers contributions of mprF, cls2, and saeR mutations to the adaptation and resistance mechanisms of S. aureus against daptomycin. These findings enrich our understanding of how S. aureus acquired resistance to daptomycin, thus paving the way for the development of more effective treatment strategies and offering potential molecular markers for resistance surveillance.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is a rarely reported lineage worldwide. This study aimed to trace the dissemination of the emerging MRSA ST630 clones in China and investigate their virulence potential. We collected 22 ST630-MRSA isolates from across China and performed whole-genome sequencing analysis and virulence characterization on these isolates. Epidemiological results showed that MRSA ST630 isolates were primarily isolated from pus/wound secretions, mainly originating from Jiangxi province, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type V (SCCmec V) predominated (11/22, 50.0%) among the MRSA ST630 isolates. Interestingly, nearly half (45.5%) of the 22 ST630-MRSA isolates tested lacked intact SCCmec elements. Phylogenetic analysis demonstrated that ST630-MRSA could be divided into two distinct clades, with widespread dissemination mainly in Chinese regions. Five representative isolates were selected for phenotypic assays, including hemolysin activity, real-time fluorescence quantitative PCR, western blot analysis, hydrogen peroxide killing assay, blood killing assay, cell adhesion and invasion assay, and mouse skin abscess model. The results showed that, compared to the USA300-LAC strain, ST630 isolates exhibited particularly strong invasiveness and virulence in the aforementioned phenotypic assays. This study described the emergence of a highly virulent ST630-MRSA lineage and improved our insight into the molecular epidemiology of ST630 clones in China.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is an emerging clone with an increasing isolation rate in China. This study raises awareness of the hypervirulent MRSA ST630 clones in China and alerts people to their widespread dissemination. ST630-staphylococcal cassette chromosome mec V is a noteworthy clone in China, and we present the first comprehensive genetic and phenotypic analysis of this lineage. Our findings provide valuable insights for the prevention and control of infections caused by this emerging MRSA clone.

ABSTRACT Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

The World Health Organization has identified high-priority target product profiles for new TB diagnostics which include rapid biomarker-based, non-sputum-based diagnostic testing, using an easily accessible sample. The Cepheid 3-gene Host Response Fingerstick Blood Prototype Test (MTB-HR) quantifies relative mRNA levels of a 3-gene signature (GBP5, DUSP3, and KLF2) from a whole-blood sample on the GeneXpert platform. The objective of the present study was to evaluate the performance of the MTB-HR to distinguish between active tuberculosis (ATB), latent Mycobacterium tuberculosis infection (LTBI), other pulmonary diseases, and healthy volunteers at a tertiary care centre. Among 653 participants enrolled in this study, 192 were diagnosed as having ATB, and the remaining 461 were classified as non-ATB, including 137 cases of LTBI, 224 cases of other pulmonary diseases, and 100 healthy volunteers. The corresponding AUCs of the MTB-HR in distinguishing untreated ATB from non-ATB, LTBI, other pulmonary diseases, and healthy volunteers were 0.814 (95% CI, 0.760-0.868, sensitivity 76.1%, specificity 71.6%), 0.739 (95% CI, 0.667-0.812, sensitivity 59.7%, specificity 78.1%), 0.825 (95% CI, 0.770-0.880, sensitivity 82.1%, specificity 65.6%), 0.892 (95% CI, 0.839-0.945, sensitivity 76.1%, specificity 88.0%), respectively. When only samples with TAT of less than 1 h were included, the AUC of the MTB-HR in distinguishing untreated ATB from non-ATB was largest, 0.920 (95% CI, 0.822-1.000, sensitivity 81.3%, specificity 87.7%). In conclusion, the MTB-HR assay shows potential as a rapid, blood-based screening and triage test for ATB, especially for untreated ATB, with the advantage of increased diagnostic yield since blood is more readily available.

ABSTRACTPrevious studies have shown that the increased prevalent ST764 clone in China, Japan, and other Asian areas. However, the knowledge of the genetic features and virulence characteristics of methicillin-resistant Staphylococcus aureus (MRSA) ST764 in China is still limited. In this study, we identified 52 ST764-SCCmec type II isolates collected from five cities in China between 2014 and 2021. Whole genome sequencing showed that the most common staphylococcal protein A (spa) types of ST764 in China were t002 (55.78%) and t1084 (40.38%). Virulence assays showed that ST764-t1084 isolates had high haemolytic activity and α-toxin levels. Of the critical regulatory factors affecting α-toxin production, only the SaeRS was highly expressed in ST764-t1084 isolates. Mouse abscess model indicated that the virulence of ST764-t1084 isolates was comparable to that of S. aureus USA300-LAC famous for its hypervirulence. Interestingly, ST764-t002 isolates exhibited stronger biofilm formation and cell adhesion capacities than ST764-t1084 isolates. This seems to explain why ST764-t002 subclone has become more prevalent in China in recent years. Phylogenetic analysis suggested that all ST764 isolates from China in Clade III were closely related to KUN1163 (an isolate from Japan). Notably, genomic analysis revealed that the 52 ST764 isolates did not carry arginine catabolic mobile element (ACME), which differed from ST764 isolates in Japan. Additionally, most ST764 isolates (69.23%) harboured an obvious deletion of approximately 5 kb in the SCCmec II cassette region compared to KUN1163. Our findings shed light on the potential global transmission and genotypic as well as phenotypic characteristics of ST764 lineage.

ABSTRACT Patients with Mycobacterium intracellulare pulmonary disease are more likely to experience poor treatment outcomes if they have been observed with microbiological persistence after 6 months of treatment. This study aims to identify the risk factors for microbiological persistence and describe the changes in the minimum inhibitory concentration (MIC) during antimycobacterial treatment. This retrospective case-control study enrolled patients diagnosed with M. intracellulare pulmonary disease between April 2017 and September 2021 at Shanghai Pulmonary Hospital. Patients with positive cultures after 6 months of treatment (positive group) were matched by age and sex in a 1:1 ratio to patients with negative conversion (negative group). Totally, 46 pairs of patients were analyzed. Risk factors for microbiological persistence at month 6 were smoking, previous tuberculosis treatment, chronic lung diseases, a positive baseline acid-fast bacilli smear, and adverse drug reactions; the risk was reduced by a regimen containing ethambutol, ≥3 effective drugs, and a higher pre-treatment absolute lymphocyte count. Regarding the drug-resistance profile, the negative group had a higher proportion of susceptibility to clarithromycin (100.0% vs 84.8%, P = 0.012). Most isolates were susceptible or intermediate to amikacin in both groups (93.5% and 84.8%, respectively). Nine patients (16.4%, 9/55) had a change in the drug-resistance profile, including four who changed from clarithromycin susceptible to clarithromycin resistant, and the other three reversed. Two pairs of isolates had a change in resistance to amikacin. In conclusion, risk factors for microbiological persistence were identified, and the change in MIC values during antimycobacterial treatment indicated the need for monitoring to enable timely adjustment of the regimen. IMPORTANCE Nontuberculous mycobacteria pulmonary disease (NTM-PD) has been recognized as an important public health issue because of its increasing incidence globally, low cure rate, and high recurrence rate. NTM-PD has innate resistance to many first-line anti-tuberculous drugs, which limits the treatment options. Mycobacterium intracellulare is reportedly the most important pathogenic NTM and accounts for the highest proportion of NTM-PD in China. A previous study suggested that poor microbiological response after 6 months of treatment is predictive of treatment failure. The present study investigated the risk factors associated with persistent positive sputum cultures by treatment month 6 in patients with M. intracellulare pulmonary disease and the variation in minimum inhibitory concentration patterns in clinical settings. This information might help to identify patients at higher risk of treatment failure and enable the timely provision of necessary interventions.

The increasing prevalence of multidrug-resistant (MDR) Gram-negative bacteria and comparatively limited options of antibiotics pose a major threat to public health worldwide. Polymyxin B is the last resort against extensively resistant Gram-negative bacterial infections. However, a large number of Gram-negative bacteria exhibited high-level resistance to Polymyxin B, bringing challenges for antimicrobial chemotherapy. Combination therapies using polymyxins and other antibiotics are recommended to treat multidrug-resistant pathogens. In this study, we selected Gram-negative bacterial strains, including Klebsiella pneumoniae and Escherichia coli, to explore whether fusidic acid and polymyxin B have a synergistic killing effect. Through broth microdilution, we observed that minimum inhibitory concentrations (MICs) against polymyxin B in the isolates tested were significantly reduced by the addition of fusidic acid. Notably, chequerboard analysis indicated a synergistic effect between polymyxin B and fusidic acid. In addition, subsequent time-kill experiments showed that the combination of polymyxin B and fusidic acid was more effective than a single drug in killing bacteria. Finally, our investigation utilizing the murine model revealed a higher survival rate in the combination therapy group compared to the monotherapy group. Our research findings provide evidence of the synergistic effect between polymyxin B and fusidic acid. Fusidic acid was shown to increase the sensitivity of multi-drug resistant E. coli and K. pneumoniae to polymyxin B, thereby enhancing its bactericidal activity. This study provides new insights into a potential strategy for overcoming polymyxin B resistance, however, further investigations are required to evaluate their feasibility in real clinical settings.

The aim of this study was to investigate the genomic epidemiology of MRSA in China to identify predominant lineages and their associated genomic and phenotypic characteristics. In this study, we conducted whole-genome sequencing on 565 MRSA isolates from 7 provinces and municipalities of China between 2014 and 2020. MRSA isolates were subjected to MLST, spa typing, SCCmec typing, analysis of virulence determinants and antimicrobial susceptibility testing. Among 565 MRSA isolates tested, clonal complex (CC) 59 (31.2%), CC5 (23.4%) and CC8 (13.63%) were the major lineages, and the clonal structure was dominated by ST59-t437-IV (14.9%), ST239-t030-III (6.4%) and ST5-t2460-II (6.0%), respectively. Of note, CC8, the predominant lineage in 2014–2015, was replaced by CC59 after 2016. Interestingly, the extension and unstable structure of the CC5 population was observed, with ST5-t311-II, ST764-t1084-II, ST5-t2460-II and ST764-t002-II existing complex competition. Further analysis revealed that virulence determinant profiles and antibiograms were closely associated with the clonal lineage. The CC59 MRSA was less resistant to most tested antimicrobials and carried fewer resistance determinants. But rifampicin resistance and mupirocin resistance were closely linked with CC8 and CC5, respectively. MRSA isolates conservatively carried multiple virulence genes involved in various functions. PVL encoding genes were more common in ST338, CC30, CC398, ST8 and CC22, while tsst-1 was associated with ST5. In conclusion, the community-associated CC59-ST59-t437-IV lineage was predominant in China, with diverse clonal isolates alternately circulating in various geographical locations. Our study highlights the need for MRSA surveillance in China to monitor changes in MRSA epidemiology.

Methicillin-resistant Staphylococcus aureus (MRSA) ST8 strains have spread worldwide, causing outbreaks in various regions. However, this clone has only been sporadically reported in China. Consequently, detailed information regarding the phylogeny and potential virulence of S. aureus ST8 strains in China remains unknown. In this study, we characterized six ST8 strains collected from three tertiary hospitals in China, including three MRSA (MR50, MR526, and MR254) and three MSSA (H78, H849 and H863). Whole genome sequencing and phylogenetic analysis showed that the six strains formed two separate clusters, including two (MR50 and MR526) and four (MR254, H78, H849 and H863) isolates, respectively. Among them, MR50 and MR526 harboured spa t008, SCCmec IVa, arginine catabolic mobile element, and were phylogenetically close to the epidemic USA300 strains, while other four strains belonged to spa t9101 and formed a unique branch. MR254 carried a novel hybrid SCCmec element (namely SCCmec254). Same as the USA300 prototype strain LAC, the China S. aureus ST8 strains produced weak biofilms except MR254. Among them, MR254 had significantly stronger haemolysis ability and higher α-toxin levels than others, while MR526 showed comparable haemolysis and α-toxin production levels as USA300-LAC. In mouse skin abscess model, MR254 showed particularly strong invasions, accompanied by necrosis, while MR526 exhibited similar infection levels as USA300-LAC. These data suggested that the China MRSA ST8 isolates (e.g. MR254 and MR526) were highly virulent, displaying higher or similar virulence potential as the epidemic USA300 strain. Active surveillance should be enacted to closely monitor the further spread of these hyper-virulent MRSA strains in China.

Major Histocompatibility Complex Class II (MHC II) deficiency is a rare primary immunodeficiency disorder (PID) with autosomal recessive inheritance pattern. The outcome is almost fatal owing to delayed diagnosis and lacking of effective therapy. Therefore, prompt diagnosis, timely and effective treatment are critical. Here, we report a 117-day-old boy with diarrhea, cough, cyanosis and tachypnea who was failed to be cured by empiric antimicrobial therapy initially and progressed to severe pneumonia and respiratory failure. The patient was admitted to the pediatric intensive care unit (PICU) immediately and underwent a series of tests. Blood examination revealed elevated levels of inflammatory markers and cytomegalovirus DNA. Imaging findings showed signs of severe infection of lungs. Finally, the diagnosis was obtained mainly through next-generation sequencing (NGS). We found out what pathogenic microorganism he was infected via repeated conventional detection methods and metagenomic next-generation sequencing (mNGS) of sputum and bronchoalveolar lavage fluid (BALF). And his whole exome sequencing (WES) examination suggested that CIITA gene was heterozygous mutation, a kind of MHC II deficiency diseases. After aggressive respiratory support and repeated adjustment of antimicrobial regimens, the patient was weaned from ventilator on the 56th day of admission and transferred to the immunology ward on the 60th day. The patient was successful discharged after hospitalizing for 91 days, taking antimicrobials orally to prevent infections post-discharge and waiting for stem cell transplantation. This case highlights the potential importance of NGS in providing better diagnostic testing for unexplained infection and illness. Furthermore, pathogens would be identified more accurately if conventional detection techniques were combined with mNGS.

Suppressing shock-induced flow separation has been a long-standing problem in the design of supersonic vehicles. To reduce the structural and design complexity of control devices, a passive control technique based on micro-serrations is proposed and its controlling effects are preliminarily investigated under test conditions in which the Mach number is 2.5 and the ramp creating an incident shock is 15 deg. Meanwhile, a vorticity-based criterion for assessing separation scales is developed to resolve the inapplicability of the zero skin friction criterion caused by wall unevenness. The simulations demonstrate that the height of the first stair significantly influences the separation length. Generally, the separation length is shorter at higher stairs, but when the height is greater than half of the thickness of the incoming boundary layer, the corresponding separation point moves upstream. A stair with a height of only 0.4 times the thickness of the boundary layer reduces the separation length by 2.69%. Further parametric analysis reveals that while the remaining serrations have limited effects on the flow separation, an optimization of their shape (depth and width) can create more favorable spanwise vortices and offer a modest improvement of the overall controlling performance. Compared to the plate case, a 9.13% reduction in the separation length can be achieved using a slightly serrated design in which the leading stair is 0.1 high and the subsequent serrations are 0.2 deep and 0.05 wide (nondimensionalized, with the thickness of the incoming boundary layer). Meanwhile, the micro-serration structure even brings less drag. Considering the minor modification to the structure, the proposed method has the potential for use in conjunction with other techniques to exert enhanced control on separations.

ABSTRACT: Objectives: To evaluate the performance of nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in predicting the drug resistance of Mycobacterium tuberculosis. Methods: A total of 115 rifampin-resistant and 53 rifampin-susceptible tuberculosis (TB) clinical isolates were randomly selected from TB strains stored at -80℃ in the clinical laboratory of Shanghai Pulmonary Hospital. Nucleotide MALDI-TOF-MS was performed to predict the drug resistance using phenotypic susceptibility as the gold standard. Results: The overall assay sensitivities and specificities of nucleotide MALDI-TOF-MS were 92.2% and 100.0% for rifampin, 90.9% and 98.6% for isoniazid, 71.4% and 81.2% for ethambutol, 85.1% and 93.1% for streptomycin, 94.0% and 100.0% for amikacin, 77.8% and 99.3% for kanamycin, 75.0% and 93.3% for ofloxacin, and 75.0% and 93.3% for moxifloxacin. The concordances between nucleotide MALDI-TOF-MS antimicrobial susceptibility testing (AST) and phenotypic AST were 94.6% (rifampin), 90.1% (isoniazid), 79.2% (ethambutol), 89.9% (streptomycin), 99.4% (amikacin), 97.0% (kanamycin), 88.1% (ofloxacin), and 88.0% (moxifloxacin). Conclusion: Nucleotide MALDI-TOF-MS could be a promising tool for rapid detection of Mycobacterium tuberculosis drug sensitivity to rifampin, isoniazid, ethambutol, streptomycin, amikacin, kanamycin, ofloxacin, and moxifloxacin.

Carbapenem-resistant Enterobacterales (CRE) infection is highly endemic in China; Klebsiella pneumoniae carbapenemase (KPC) 2–producing CRE is the most common, whereas KPC-3–producing CRE is rare. We report an outbreak of KPC-3–producing Enterobacterales infection in China. During August 2020–June 2021, 25 blaKPC-3–positive Enterobacteriale isolates were detected from 24 patients in China. Whole-genome sequencing analysis revealed that the blaKPC-3 genes were harbored by IncX8 plasmids. The outbreak involved clonal expansion of KPC-3–producing Serratia marcescens and transmission of blaKPC-3 plasmids across different species. The blaKPC-3 plasmids demonstrated high conjugation frequencies (10−3 to 10−4). A Galleria mellonella infection model showed that 2 sequence type 65 K2 K. pneumoniae strains containing blaKPC-3 plasmids were highly virulent. A ceftazidime/avibactam in vitro selection assay indicated that the KPC-3–producing strains can readily develop resistance. The spread of blaKPC-3–harboring IncX8 plasmids and these KPC-3 strains should be closely monitored in China and globally.

ABSTRACT ST22 MRSA (methicillin-resistant Staphylococcus aureus) strains are only sporadically reported in China. Through the phylogenetic reconstruction of 30 ST22 strains from China and 480 ST22 strains from global sources, we found that the global ST22 strains can be divided into three clades (I, II, and III). The China ST22 strains were found primarily in clade II (IIb and IIc) and also in clade III, indicating that the China ST22-MRSA clones have different origins. The China subclade IIb strains (SCCmec Vb-t309) may evolve from the native ST22 MSSA clone, while the China IIc strains may have spread from other countries. Subclade IIc (SCCmecIVa-t309) strains exhibited particularly strong lethality and invasiveness in Galleria mellonella infection and mouse skin abscess models in comparison to USA300 and other dominant China HA-MRSA (ST5 and ST239) or CA-MRSA (ST59) strains. This study described the emergence of a highly virulent ST22 MRSA subclade and improved our insight into the molecular epidemiology of ST22 strains in China. IMPORTANCE ST22 is a successful hospital-associated MRSA lineage which first appeared in the United Kingdom as EMRSA-15. At present, ST22 MRSA clones are spreading rapidly around the world and even replaced other dominant clones in some regions. We placed the Chinese ST22 in the worldwide phylogeny of ST22, demonstrating a distinctive molecular epidemiology and to our knowledge, this is the first time that a novel clade of ST22 has been found in China. Among the 15 ST22 MRSA strains belonging to the novel clade, 14 ST22 SCCmecIVa strains from different regions carried both pvl and tst and displayed significantly higher in vitro and in vivo virulence in comparison to other clade/subclade ST22 strains as well as other common China HA-MRSA or CA-MRSA strains. The further spread of this subclade of strains could pose a serious threat to the health system in China and other regions.

ABSTRACT Staphylococcus aureus is an important human pathogen and brings about many community-acquired, hospital-acquired, and biofilm-associated infections worldwide. It tends to form biofilms, triggering the release of toxins and initiating resistance mechanisms. As a result of the development of S. aureus tolerance to antibiotics, there are few drugs can availably control biofilm-associated infections. In this study, we synthesized a novel small-molecule compound CY-158-11 (C22H14Cl2NO2Se2) and proved its inhibitory effect on the biofilm formation of S. aureus at a subinhibitory concentration (1/8 MIC). The subinhibitory concentration of CY-158-11 not only did not affect the growth of bacteria but also had no toxicity to A549 cells or G. mellonella. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed by SYTO 9 and PI staining through confocal laser scanning microscopy. Moreover, CY-158-11 effectively prevented initial attachment and repressed the production of PIA instead of autolysis. RT-qPCR analysis also exhibited significant suppression of the genes involved in biofilm formation. Taken together, CY-158-11 exerted its inhibitory effects against the biofilm formation in S. aureus by inhibiting cell adhesion and the expression of icaA related to PIA production. IMPORTANCE Most bacteria exist in the form of biofilms, often strongly adherent to various surfaces, causing bacterial resistance and chronic infections. In general, antibacterial drugs are not effective against biofilms. The small-molecule compound CY-158-11 inhibited the biofilm formation of S. aureus at a subinhibitory concentration. By hindering adhesion and PIA-mediated biofilm formation, CY-158-11 exhibits antibiofilm activity toward S. aureus. These findings point to a novel therapeutic agent for combating intractable S. aureus-biofilm-related infections.

BackgroundAnemia leads to a lower cure rate and poor prognosis in tuberculosis patients. Effective predictors for the prognosis of tuberculosis with anemia (A-TB) are urgently needed. Monocyte has been proven to be a prognostic biomarker of many lung diseases. Whether monocyte that the predominant innate immune cell as early defense against tuberculosis can predict A-TB is not known.MethodsData for A-TB patients with initial treatment in Shanghai Pulmonary Hospital were retrospectively collected and analyzed. Logistics regression analysis was used to study the correlation between peripheral blood cells and treatment outcomes. The receiver operating characteristic (ROC) curve was used to determine the cut-off value. We estimated a 12-month prognosis using Kaplan–Meier techniques. The Cox proportional hazards model was used for the univariate and multivariate analyses to analyze the predictors of poor prognosis of A-TB.ResultsOf 181 patients analyzed, 94 were cured and 87 non-cured. Logistic regression analysis identified monocyte as an independent immune-related risk factor for the prognosis of A-TB (OR: 7.881, 95% CI: 1.675–37.075, P = 0.009). The ROC curve analysis proved that the most discriminative cut-off value of monocyte was 0.535 × 10^9/L. K–M analysis demonstrated that the cumulative cure rates of A-TB were significantly higher in A-TB with monocyte < 0.535 × 10^9/L (69.62%) than that in those with monocyte ≥ 0.535 × 10^9/L (38.24%) (Log-rank, χ2 = 16.530, P < 0.0001). On univariate and multivariable analysis, monocyte was an independent predictor of poor prognosis in A-TB. Similarly, monocyte was also an independent predictor of poor pulmonary cavity closure in A-TB (HR: 3.614, 95% CI: 1.335–9.787, P = 0.011).ConclusionIn A-TB patients, elevated monocyte was associated with poor prognosis and poor cavity pulmonary closure. Monocyte may provide a simple and inexpensive prognostic biomarker in A-TB.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 45 (ST45) was rarely found in China. This study was conducted to trace the transmission and evolution of emerging MRSA ST45 strains in mainland China and explore its virulence. A total of 27 ST45 isolates were included for whole-genome sequencing and genetic characteristic analysis. Epidemiological results showed that MRSA ST45 isolates were often obtained from blood, primarily originated in Guangzhou, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type IV (SCCmec IV) dominated in MRSA ST45 (23/27, 85.2%). ST45-SCCmec V was located on a phylogenetic clade distinct from the SCCmec IV cluster. We selected two representative isolates, MR370 (ST45-SCCmec IV) and MR387 (ST45-SCCmec V), and performed hemolysin activity, a blood killing assay, a Galleria mellonella infection model, and a mouse bacteremia model, as well as real-time fluorescence quantitative PCR. MR370 was proved to have extreme virulence in the phenotypic assays and at the mRNA level compared with ST59, ST5, and USA300 MRSA strains. MR387 was comparable to USA300-LAC on the phenotype and was verified to have higher expression of scn, chp, sak, saeR, agrA, and RNAIII than USA300-LAC. The results emphasized the extraordinary performance of MR370 and the good potential of MR387 in virulence causing bloodstream infection. Meanwhile, we conclude that China MRSA ST45 showed two different clonotypes, which may be widespread in the future. The entire study is valuable as a timely reminder and reports virulence phenotypes of China MRSA ST45 for the first time. IMPORTANCE Methicillin-resistant Staphylococcus aureus ST45 is epidemic worldwide. This study contributed to the awareness of the Chinese hyper-virulent MRSA ST45 strains and served as a timely reminder of its wide dissemination of clonotypes. Further, we provide novel insights for prevention from the perspective of bloodstream infections. ST45-SCCmec V is a clonotype deserving special attention in China, and we performed genetic and phenotypic analyses for the first time on it.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and restricted therapeutic options pose a global threat to public health. Aminoglycosides are a wise choice, which can effectively reduce the mortality rate when combined with β-lactam drugs. However, in this study, we identified a ST15-KL112 CRKP FK3006 which not only exhibited resistance to carbapenems, but also exhibited high level resistance to aminoglycosides. In addition to the multidrug resistant phenotype, FK3006 also owned typical pathogenic characteristic, including hypermucoviscosity and hypervirulence phenotypes. According to the whole-genome sequencing, one pLVPK-like virulence plasmid, and three key resistant plasmids (blaOXA-232, blaCTX-M-15, and rmtF) were observed in FK3006. Compared to other typical ST15 CRKP, the presence of pLVPK-like virulence plasmid (p3006-2) endowed the FK3006 with high virulence features. High siderophore production, more cell invasive and more resistant to serum killing was observed in FK3006. The Galleria mellonella infection model also further confirmed the hypervirulent phenotype of FK3006 in vivo. Moreover, according to the conjugation assay, p3006-2 virulence plasmid also could be induced transfer with the help of conjugative IncFIIK p3006-11 plasmid (blaCTX-M-15). In addition to the transmissible plasmid, several insertion sequences and transposons were found around blaCTX-M-15, and rmtF to generate the mobile antimicrobial resistance island (ARI), which also make a significant contribution to the dissemination of resistant determinants. Overall, we reported the uncommon co-existence of blaOXA-232, rmtF-encoding plasmids, and pLVPK-like virulence plasmid in ST15-KL112 K. pneumoniae. The dissemination threatens of these high-risk elements in K. pneumoniae indicated that future studies are necessary to evaluate the prevalence of such isolates.

ABSTRACT Mupirocin, a topical antimicrobial agent, is an important component in the eradication of methicillin-resistant Staphylococcus aureus (MRSA) colonization. The molecular characteristics of 46 mupirocin-resistant MRSA (MR-MRSA) clinical isolates were analyzed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec element (SCCmec) typing, spa typing, and analysis of virulence genes. All 26 MRSA isolates with low-level mupirocin resistance possessed a V588F mutation in ileS. Among 20 MRSA isolates with high-level resistance to mupirocin, all carried mupA; 2 isolates also possessed the V588F mutation in ileS, and 1 possessed the V631F mutation in ileS (isoleucyl-tRNA synthetase). The majority of MR-MRSA isolates were resistant to erythromycin, clindamycin, tetracycline, ciprofloxacin, and gentamicin, but the rates of resistance to rifampin and fusidic acid were 8.7% and 6.5%, respectively. Eight sequence types (STs) were found among the 46 MR-MRSA isolates, of which ST764 was the most prevalent (76.1%). The most frequent spa type identified was t1084 (52.2%). The SCCmec type most frequently found was type II (80.4%). The most common clone among low-level MR-MRSA isolates was ST764-MRSA-SCCmec type II-t1084 (23 isolates), while ST764-MRSA-SCCmec type II-t002 (9 isolates) was the most common clone among high-level MR-MRSA isolates. Additionally, all toxin genes except the seb gene were not identified among ST764 isolates. Among clonal complex 5 (CC5) isolates, immune evasion cluster (IEC)-associated genes (chp, sak, and scn) and seb were present in ST764 but absent in ST5, while sec, sel1, tsst-1, and hlb genes were identified in ST5 but absent in ST764. In conclusion, the spread of CC5 clones, especially a novel ST764-MRSA-SCCmec type II-t1084 clone with high-level resistance to mupirocin, was responsible for the increase in mupirocin resistance. These findings indicated that the emergence of the ST764 MR-MRSA clone involves a therapeutic challenge for treating serious MRSA infections. IMPORTANCE Mupirocin, a topical antibiotic that is commonly used for the nasal decolonization of MRSA and methicillin-sensitive Staphylococcus aureus in hospital settings and nursing homes, was introduced as a highly effective antibiotic against MRSA. Mupirocin acts by competitively binding isoleucyl-tRNA synthetase, thereby disrupting protein synthesis. This drug shows bacteriostatic and bactericidal activity at low and high concentrations, respectively. However, with the increase in mupirocin use, low-level and high-level resistance during nasal mupirocin treatment has been reported. In a previous study, the proportion of MRSA strains with high-level mupirocin resistance in a Canadian hospital increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% (2000 to 2004).

ObjectiveThe widespread use of antibiotics has inevitably led to the emergence of multidrug-resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), making treatment of this infection a serious challenge. This study aimed to explore new treatment strategies for MRSA infection.MethodsThe structure of Fe3O4 NPs with limited antibacterial activity was optimized, and the Fe2+ ↔ Fe3+ electronic coupling was eliminated by replacing 1/2 Fe2+ with Cu2+. A new type of copper-containing ferrite nanoparticles (hereinafter referred to as Cu@Fe NPs) that fully retained oxidation–reduction activity was synthesized. First, the ultrastructure of Cu@Fe NPs was examined. Then, antibacterial activity was determined by testing the minimum inhibitory concentration (MIC) and safety for use as an antibiotic agent. Next, the mechanisms underlying the antibacterial effects of Cu@Fe NPs were investigated. Finally, mice models of systemic and localized MRSA infections was established for in vivo validation.ResultsIt was found that Cu@Fe NPs exhibited excellent antibacterial activity against MRSA with MIC of 1 μg/mL. It effectively inhibited the development of MRSA resistance and disrupted the bacterial biofilms. More importantly, the cell membranes of MRSA exposed to Cu@Fe NPs underwent significant rupture and leakage of the cell contents. Cu@Fe NPs also significantly reduced the iron ions required for bacterial growth and contributed to excessive intracellular accumulation of exogenous reactive oxygen species (ROS). Therefore, these findings may important for its antibacterial effect. Furthermore, Cu@Fe NPs treatment led to a significant reduction in colony forming units within intra-abdominal organs, such as the liver, spleen, kidney, and lung, in mice with systemic MRSA infection, but not for damaged skin in those with localized MRSA infection.ConclusionThe synthesized nanoparticles has an excellent drug safety profile, confers high resistant to MRSA, and can effectively inhibit the progression of drug resistance. It also has the potential to exert anti-MRSA infection effects systemically in vivo. In addition, our study revealed a unique multifaceted antibacterial mode of Cu@Fe NPs: (1) an increase in cell membrane permeability, (2) depletion of Fe ions in cells, (3) generation of ROS in cells. Overall, Cu@Fe NPs may be potential therapeutic agents for MRSA infections.

Summary: Background: Mobile plasmids play a key role in spurring the global dissemination of multidrug-resistant (MDR) K. pneumoniae, while plasmid curing has been recognized as a promising strategy to combat antimicrobial resistance. Here we exploited a K. pneumoniae native CRISPR system to cure the high-risk IncFII plasmids. Methods: We examined matched protospacers in 725 completely sequenced IncFII plasmids from K. pneumoniae genomes. Then, we re-engineered a native CRISPR-Cas3 system and deliver the CRISPR-Cas3 system via conjugation. Plasmid killing efficiency and G. mellonella infection model were applied to evaluate the CRISPR-Cas3 immunity in vitro and in vivo. Findings: Genomic analysis revealed that most IncFII plasmids could be targeted by the native CRISPR-Cas3 system with multiple matched protospacers, and the targeting regions were highly conserved across different IncFII plasmids. This conjugative endogenous CRISPR-Cas3 system demonstrated high plasmid curing efficiency in vitro (8-log decrease) and in vivo (∼100% curing) in a Galleria mellonella infection model, as well as provided immunization against the invasion of IncFII plasmids once the system entering a susceptible bacterial host. Interpretation: Overall, our work demonstrated the applicability of using native CRISPR-mediated plasmid curing to re-sensitize drug-resistant K. pneumoniae to multiple antibiotics. This work provided strong support for the idea of utilizing native CRISPR-Cas systems to tackle AMR in K. pneumoniae. Funding: This work was supported by research grants National Natural Science Foundation of China [grant numbers 81871692, 82172315, 82102439, and 82202564], the Shanghai Science and Technology Commission [grant number 19JC1413002], and Shanghai Sailing Program [grant number 22YF1437500].

ABSTRACT Recently, the incidence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing worldwide, especially in immunocompromised patients and those with potential chronic lung disease. Vitek MS v3.0 matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and reliable method for identifying mycobacteria in clinical laboratories. This study aimed to evaluate the performance of Vitek MS v3.0 by isolating NTM directly from automated liquid medium systems using patient samples. A total of 855 Mycobacterium growth indicator tube (MGIT)-positive liquid cultures were investigated. Among them, 658 (77.0%) liquid cultures were correctly identified to the species, group, or complex level, 192 (23.0%) resulted in no identification, and 5 (0.6%) were misidentified at the species level. DNA sequencing identified 855 NTM isolates from liquid cultures, comprising 316 isolates of rapidly growing mycobacteria (RGM) and 539 isolates of slow-growing mycobacteria (SGM). Using the Vitek MS system, the RGM integral identification rate (276/316 [87.34%]) was higher than the SGM rate (381/539 [70.69%]) (P

Multi-drug resistant Staphylococcus aureus infection is still a serious threat to global health. Therefore, there is an urgent need to develop new antibacterial agents based on virulence factor therapy to overcome drug resistance. Previously, we synthesized SYG-180-2-2 (C21H16N2OSe), an effective small molecule compound against biofilm. The aim of this study was to investigate the anti-virulence efficacy of SYG-180-2-2 against Staphylococcus aureus. MIC results demonstrated no apparent antibacterial activity of the SYG-180-2-2. The growth curve assay showed that SYG-180-2-2 had nonlethal effect on S. aureus. Besides, SYG-180-2-2 strongly inhibited the hemolytic activity and staphyloxanthin synthesis in S. aureus. Inhibition of staphyloxanthin by SYG-180-2-2 enhanced the sensitivity of S. aureus to oxidants and human whole blood. In addition, SYG-180-2-2 significantly decreased the expression of saeR-mediated hemolytic gene hlb and staphyloxanthin-related crtM, crtN and sigB genes by quantitative polymerase chain reaction (qPCR). Meanwhile, the expression of oxidative stress-related genes sodA, sodM and katA also decreased. Galleria Mellonella assay revealed that SYG-180-2-2 was not toxic to larvae. Further, the larvae infection model showed that the virulence of bacteria was significantly reduced after 4 μg/mL SYG-180-2-2 treatment. SYG-180-2-2 also reduced skin abscess formation in mice by reducing bacterial burden and subcutaneous inflammation. In conclusion, SYG-180-2-2 might be a promising agent to attenuate the virulence of S. aureus by targeting genes associated with hemolytic activity and staphyloxanthin synthesis.

Abstract Background To verify the efficacy and safety of an inexpensive standardized regimen for multidrug-resistant tuberculosis (MDR-TB) with low resistance to isoniazid (INH), a multicenter prospective study was conducted in eastern China. Methods Patients diagnosed as MDR-TB with low concentration INH resistance and rifampicin resistance, second-line/injectable agents sensitive were prospectively enrolled, given the regimen of Amikacin (Ak)–Fluoroquinolones (FQs)–Cycloserine (Cs)–Protionamide (Pto)–PasiniaZid (Pa)–Pyrazinamide (Z) for 6 months followed by 12 months of FQs–Cs–Pto–Pa–Z, and then followed up for treatment outcomes and adverse events (AEs). Results A total of 114 patients were enrolled into the study. The overall favorable treatment rate was 79.8% (91/114). Among 91 cases with favorable treatment, 75.4% (86/114) were cured and 4.4% (5/114) were completed treatment. Regarding to unfavorable outcomes, among 23 cases, 8.8% (10/114) had failures, 8.8% (10/114) losing follow up, 0.9% (1/114) had treatment terminated due to intolerance to drugs and 1.8% (2/114) died. Treatment favorable rate was significantly higher in newly treated MDR-TB (91.7%, 33/36) than that in retreated MDR-TB (74.4%, 58/78, p 0.03). The investigators recorded 42 AEs occurrences in 30 of 114 patients (26.3%). Clinicians rated most AEs as mild or moderate (95.24%, 40/42). Conclusions The regimen was proved to be effective, safe and inexpensive. It is suitable for specific drug resistant population, especially for newly-treated patients, which could be expected to be developed into a short-course regimen. Clinical trials registration China Clinical Trial Registry ChiCTR-OPC-16009380

ABSTRACT The human fungal pathogen Candida glabrata is phylogenetically closely related to Saccharomyces cerevisiae, a model eukaryotic organism. Unlike S. cerevisiae, which has both haploid and diploid forms and a complete sexual cycle, C. glabrata has long been considered a haploid and asexual species. In this study, we analyzed the ploidy states of 500 clinical isolates of C. glabrata from four Chinese hospitals and found that approximately 4% of the isolates were in or able to spontaneously switch to an aneuploid (genomic DNA, 1N-2N), diploid (2N), or hyperdiploid (>2N) form under in vivo or in vitro conditions. Stable diploid cells were identified in 3% of the isolates (15/500). Of particular interest, one clinical strain existed only in the diploid form. Multilocus sequence typing (MLST) assays revealed two major genetic clusters (A and B) of C. glabrata isolates. Most of the isolates (70%) from China belonged to the A cluster, whereas most of the isolates from other countries (such as Iran, Japan, United States, and European countries) belonged to the B cluster. Further investigation indicated that C. glabrata cells of different ploidy forms differed in a number of respects, including morphologies, antifungal susceptibility, virulence, and global gene expression profiles. Additionally, C. glabrata could undergo spontaneous switching between the diploid and haploid forms under both in vitro and in vivo conditions. Given the absence of an apparent sexual phase, one would expect that the ploidy shifts could function as an alternative strategy that promotes genetic diversity and benefits the ability of the fungus to rapidly adapt to the changing environment. IMPORTANCE The human fungal pathogen Candida glabrata has long been thought to be a haploid organism. Here, we report the population structure and ploidy states of 500 clinical isolates of C. glabrata from China. To our surprise, we found that the ploidy of a subset of clinical isolates varied dramatically. Some isolates were in or able to switch to an aneuploid, diploid, or hyperdiploid form. C. glabrata cells with different ploidy differed in a number of biological respects, including morphology, antifungal susceptibility, virulence, and global gene expression profile. Given the absence of an apparent sexual phase in this fungus, we propose that ploidy switching could be a strategy for rapid adaptation to environmental changes and could function as an alternative to sexual reproduction.

ABSTRACT Members of the Mycobacterium abscessus complex (MABC) are multidrug-resistant nontuberculous mycobacteria and increasingly cause opportunistic pulmonary infections. However, the genetic typing of MABC isolates remains largely unclear in China. Genomic analyses were conducted for 69 MABC clinical isolates obtained from patients with lower respiratory tract infections in Shanghai Pulmonary Hospital between 2014 and 2016. The draft genomes of the 69 clinical strains were assembled, with a total length of 4.5 to 5.6 Mb, a percent GC content (GC%) ranging from 63.9 to 68.1%, and 4,492 to 5,404 genes per genome. Susceptibility test shows that most isolates are resistant to many antimicrobials, including clarithromycin, but susceptible to tigecycline. Analyses revealed the presence of genes conferring resistance to antibiotics, including macrolides, aminoglycosides, rifampicin, and tetracyclines. Furthermore, 80 to 114 virulence genes were identified per genome, including those related to the invasion of macrophages, iron incorporation, and avoidance of immune clearance. Mobile genetic elements, including insertion sequences, transposons, and genomic islands, were discovered in the genomes. Phylogenetic analyses of all MABC isolates with another 41 complete MABC genomes identified three clades; 46 isolates were clustered in clade I, corresponding to M. abscessus subsp. abscessus, and 25 strains belonged to existing clonal complexes. Overall, this is the first comparative genomic analysis of MABC clinical isolates in China. These results show significant intraspecies variations in genetic determinants encoding antimicrobial resistance, virulence, and mobile elements and controversial subspecies classification using current marker gene combinations. This information will be useful in understanding the evolution, antimicrobial resistance, and pathogenesis of MABC strains and facilitating future vaccine development and drug design. IMPORTANCE Over the past decade, infections by Mycobacterium abscessus complex (MABC) isolates have been increasingly reported worldwide. MABC strains often show a high incidence in cystic fibrosis (CF) patients, whereas in Asia, these strains are frequently recovered from non-CF patients with significant genomic diversity. The present work involves analyses of the antimicrobial resistance, virulence, and phylogeny of 69 selected MABC isolates from non-CF pulmonary patients in Shanghai Pulmonary Hospital by whole-genome sequencing; it represents the first comprehensive investigation of MABC strains in China at the genomic level. These findings highlight the diversity of this group of nontuberculous mycobacteria and provide a mechanistic understanding of evolution and pathogenesis, which is valuable for the development of novel and effective antimicrobial therapies for deadly MABC infections in China.

Abstract Background The rate of fluoroquinolone (FQ) resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) is high. The present study aimed to investigate the distribution of fluoroquinolone resistance determinants in clinical CRKP isolates associated with bloodstream infections (BSIs). Results A total of 149 BSI-associated clinical CRKP isolates collected from 11 Chinese teaching hospitals from 2015 to 2018 were investigated for the prevalence of fluoroquinolone resistance determinants, including plasmid-mediated quinolone resistance (PMQR) genes and spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. Among these 149 clinical CRKP isolates, 117 (78.5%) exhibited resistance to ciprofloxacin. The GyrA substitutions (Ser83 → IIe/Phe) and (Asp87 → Gly/Ala) were found among 112 (75.2%) of 149 isolates, while the substitution (Ser80 → IIe) of ParC was found in 111 (74.5%) of the 149 isolates. In total, 70.5% (105/149) of the CRKP isolates had at least two mutations within gyrA as well as a third mutation in parC. No mutations in the QRDRs were found in 31 ciprofloxacin susceptible CRKP isolates. Eighty-nine (56.9%) of 149 were found to carry PMQR genes including qnrS1 (43.0%), aac(6′)-Ib-cr (16.1%), qnrB4 (6.0%), qnrB2 (2.7%), and qnrB1 (1.3%). Nine isolates contained two or more PMQR genes, with one carrying four [aac(6′)-Ib-cr, qnr-S1, qnrB2, and qnrB4]. The co-existence rate of PMQR determinants and mutations in the QRDRs of gyrA and parC reached 68.5% (61/89). Seventy-four (83.1%, 74/89) PMQR-positive isolates harbored extended-spectrum beta-lactamase (ESBL)-encoding genes. Multilocus sequence typing (MLST) analysis demonstrated that the ST11 was the most prevalent STs in our study. Conclusions Mutations in the QRDRs of gyrA and parC were the key factors leading to the high prevalence of fluoroquinolone resistance among BSI-associated CRKP. The co-existence of PMQR genes and mutations in the QRDRs can increase the resistance level of CRKP to fluoroquinolones in clinical settings. ST11 CRKP isolates with identical QRDR substitution patterns were found throughout hospitals in China.

Objective: To evaluate he diagnostic performance of the Xpert Mycobacterium tuberculosis/Rifampin (MTB/RIF) assay in bronchoalveolar lavage fluid (BALF). Methods: We retrospectively reviewed the clinical data from 671 sputum-smear negative or sputum-scarce adult patients with suspected pulmonary tuberculosis (PTB) who had an Xpert MTB/RIF assay performed on BALF. The diagnostic performance of the Xpert MTB/RIF assay, smear microscopy (SM) and MTB culture was evaluated using MTB culture or final clinical diagnosis as the reference standard. Results: Compared with MTB culture, the sensitivity and specificity were 87.8% and 72.7% for the Xpert MTB/RIF assay and 11.0% and 99.2% for SM, respectively. Compared with final diagnosis, diagnostic performance was 58.9% and 83.9% for the Xpert MTB/RIF assay, 5.0% and 98.3% for SM, and 43.3% and 100% for culture, for sensitivity and specificity respectively. The Xpert MTB/RIF assay had low specificity and high sensitivity. When very low results were re-evaluated and considered MTB-negative, the specificity increased significantly. The sensitivity remained higher than SM and was similar to that of culture. Conclusions: The Xpert MTB/RIF assay adds microbiologic evidence to clinical decisions; however, close attention should be paid to very low semi-quantitative positive results.

Abstract Background In recent years, clinical Staphylococcus aureus isolates have become highly resistant to antibiotics, which has raised concerns about the ability to control infections by these organisms. The aim of this study was to clarify the effect of a new small molecule, ZY-214-4 (C19H11BrNO4), on S. aureus pigment production. Results At the concentration of 4 μg/mL, ZY-214-4 exerted a significant inhibitory effect on S. aureus pigment synthesis, without affecting its growth or inducing a toxic effect on the silkworm. An oxidant sensitivity test and a whole-blood killing test indicated that the S. aureus survival rate decreased significantly with ZY-214-4 treatment. Additionally, ZY-214-4 administration significantly reduced the expression of a pigment synthesis-related gene (crtM) and the superoxide dismutase genes (sodA) as determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. ZY-214-4 treatment also improved the survival rate of S. aureus-infected silkworm larvae. Conclusions The small molecule ZY-214-4 has potential for the prevention of S. aureus infections by reducing the virulence associated with this bacterium.

ABSTRACT Klebsiella pneumoniae is one of the most common Gram-negative bacilli isolated from bloodstream infections worldwide, and recently an increased rate of carbapenem resistance has been reported in this pathogen. This study aims to describe the genomic characteristics of carbapenem-resistant K. pneumoniae (CRKP) isolated from patients with bacteremia in China. We analyzed 147 isolates from patients with bacteremia attended in 12 referral hospitals in China between April 2015 and November 2018. We conducted a phenotypic susceptibility evaluation and whole genome sequence analysis to characterize antimicrobial resistance profile, virulence genes, and dominant clones among CRKP. ST11 accounted for most infections (n = 98, 66.6%), followed by ST45 (n = 12, 8.2%), ST15 and ST290 (n = 8, 5.4% each). KPC (n = 98, 66.7%) and NDM (n = 27, 18.4%) are the main carbapenemases detected in the CRKP isolates. We detected yersiniabactin (n = 123, 83.7%) and aerobactin (49.9%) siderophores, and both rmpA and aerobactin genes in 21 ST11 isolates (21.43%), which are considered characteristic biomarkers of hypervirulent strains. Isolates showed high resistance rates to the β-lactams (>90%) and other antibiotics classes such as fluoroquinolones, aminoglycosides and tetracyclines (50%), but were susceptible to ceftazidime-avibactam (74.8%). In addition, we detected intra-hospital transmission of ST11 and ST45 strains in single and multiple wards in several hospitals, whereas inter-hospital transmission was relatively uncommon. In summary, we observed significantly genomic diversity of CRKP bacteremia isolates in China, although KPC-2 producing ST11 strains were found to be the most common clonal types. Reducing intra-hospital transmission remains to be the key to control CRKP caused bloodstream infections in China. IMPORTANCE K. pneumoniae is one of the most frequent Gram-negative bacilli isolated from bloodstream infections worldwide and recent studies have shown an increased rate of carbapenem resistance in China. Among carbapenem-resistant K. pneumoniae (CRKP) diverse clones have been reported, especially the high-risk clone ST11, which also exhibited a multidrug resistant phenotype. In addition to the antimicrobial resistance, previous studies have detected strains co-harboring virulent traits, highlighting the potential of transmission of both antimicrobial resistant and virulent strains. Here we studied the antimicrobial resistance profile, virulence genes and hospital transmission of CRKP from bacteremic patients in China. This study showed a high clonal diversity among CRKP, with the predominance of ST11 lineages. We detected virulence markers among multidrug resistant strains, and a high number of genetically similar isolates, suggesting intra-hospital transmission within single and multiple wards. Reducing intra-hospital transmission remains to be the key to control CRKP caused bacteremia in China.

ABSTRACT The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the comparatively limited development of new antibiotics pose a major threat to public health. Aminoglycosides are important options that can lower the mortality rate effectively in combination therapy with β-lactam agents. However, in this study, we observed two multidrug-resistant (MDR) K. pneumoniae named 1632 and 1864 that exhibited high-level resistance to both carbapenems and aminoglycosides. Through whole-genome sequencing (WGS), the unusual co-occurrence of rmtB, armA, and blaKPC-2 genes, associating with two key resistance plasmids, was observed in two isolates. Notably, we also found that the armA resistance gene and virulence factor iuc operon co-occurred on the same plasmid in K. pneumoniae 1864. Detailed comparative genetic analysis showed that all these plasmids were recognized as mobilizable plasmids, as they all carry the essential oriT site. Results of conjugation assay indicated that armA-positive plasmids in two isolates could self-transfer to Escherichia coli J53 effectively, especially, the p1864-1 plasmid, which could cotransfer hypervirulent and multidrug-resistant phenotypes to other isolates. Moreover, multiple insertion sequences (ISs) and transposons (Tns) were also found surrounding the vital resistant genes, which could even form a large antibiotic resistance island (ARI) and could stimulate mobilization of resistant determinants. Overall, we report the uncommon coexistence of armA plasmid, rmtB-blaKPC-2 plasmid, and even iuc virulence operon-encoding plasmid in K. pneumoniae isolates, which greatly increased the spread of these high-risk phenotypes and which are of great concern. IMPORTANCE Carbapenemase-producing Klebsiella pneumoniae have become a great challenge for antimicrobial chemotherapy, while aminoglycosides can lower the mortality rate effectively in combination therapy with them. Unfortunately, we isolated two K. pneumoniae from blood sample of patients that not only exhibited high-level resistance to carbapenems and aminoglycosides but also showed the unusual co-occurrence of the rmtB, armA, and blaKPC-2 genes. These elements were all located on mobile plasmids and flanked by polymorphic mobile genetic elements (MGEs). What’s worse most, we also identified a conjugative virulent MDR plasmid, coharboring multiple resistant determinants, and iuc operon, which was confirmed could transfer such high-risk phenotype to other isolates. The emergence of such conjugative virulence plasmids may promote the rapid dissemination of virulence-encoding elements among Gram-negative pathogens. This uncommon coexistence of rmtB, armA, blaKPC-2, and iuc virulence operon-encoding plasmids in K. pneumoniae, presents a huge threat to clinical treatment. Future studies are necessary to evaluate the prevalence of such isolates.

ABSTRACT Our objective was to evaluate the performance of whole-genome sequencing (WGS) from early positive liquid cultures for predicting Mycobacterium tuberculosis complex (MTBC) drug resistance. Clinical isolates were obtained from tuberculosis patients at Shanghai Pulmonary Hospital (SPH). Antimicrobial susceptibility testing (AST) was performed, and WGS from early Bactec mycobacterial growth indicator tube (MGIT) 960-positive liquid cultures was performed to predict the drug resistance using the TB-Profiler informatics platform. A total of 182 clinical isolates were enrolled in this study. Using phenotypic AST as the gold standard, the overall sensitivity and specificity for WGS were, respectively, 97.1% (89.8 to 99.6%) and 90.4% (83.4 to 95.1%) for rifampin, 91.0% (82.4 to 96.3%) and 95.2% (89.1 to 98.4%) for isoniazid, 100.0% (89.4 to 100.0%) and 87.3% (80.8 to 92.1%) for ethambutol, 96.6% (88.3 to 99.6%) and 61.8% (52.6 to 70.4%) for streptomycin, 86.8% (71.9 to 95.6%) and 95.8% (91.2 to 98.5%) for moxifloxacin, 86.5% (71.2 to 91.5%) and 95.2% (90.3 to 98.0%) for ofloxacin, 100.0% (54.1 to 100.0%) and 67.6% (60.2 to 74.5%) for amikacin, 100.0% (63.1 to 100.0%) and 67.2% (59.7 to 74.2%) for kanamycin, 62.5% (24.5 to 91.5%) and 88.5% (82.8 to 92.8%) for ethionamide, 33.3% (4.3 to 77.7%) and 98.3% (95.1 to 99.7%) for para-aminosalicylic acid, and 0.0% (0.0 to 12.3%) and 100.0% (97.6 to 100.0%) for cycloserine. The concordances of WGS-based AST and phenotypic AST were as follows: rifampin (92.9%), isoniazid (93.4%), ethambutol (89.6%), streptomycin (73.1%), moxifloxacin (94.0%), ofloxacin (93.4%), amikacin (68.7%), kanamycin (68.7%), ethionamide (87.4%), para-aminosalicylic acid (96.2%) and cycloserine (84.6%). We conclude that WGS could be a promising approach to predict MTBC resistance from early positive liquid cultures. IMPORTANCE In this study, we used whole-genome sequencing (WGS) from early positive liquid (MGIT) cultures instead of solid cultures to predict drug resistance of 182 Mycobacterium tuberculosis complex (MTBC) clinical isolates to predict drug resistance using the TB-Profiler informatics platform. Our study indicates that WGS may be a promising method for predicting MTBC resistance using early positive liquid cultures.

Abstract Background Multidrug resistant (MDR) Gram-negative bacterial infections are a serious threat to human health due to the lack of effective treatments. In this study, we selected 50 Gram-negative bacterial strains, including 26 strains of Klebsiella pneumoniae and 24 strains of Escherichia coli, to explore whether resveratrol and polymyxin B have a synergistic killing effect. Results MIC values against polymyxin B were ≥ 4 μg/mL for 44 of the strains and were 2 μg/mL for the other 6 strains. MICs against polymyxin B in the isolates tested were significantly reduced by the addition of resveratrol. The degree of decline depended on the bacteria, ranging from 1/2 MIC to 1/512 MIC, and the higher the concentration of resveratrol, the greater the decrease. Checkerboard analysis indicated a synergistic effect between resveratrol and polymyxin B; the optimal drug concentration for different bacteria was different, that of resveratrol ranging from 32 μg/mL to 128 μg/mL. Subsequent time-kill experiments showed that a combination of polymyxin B and resveratrol was more effective in killing bacteria. Conclusions Our in vitro studies have shown that resveratrol can increase the sensitivity of MDR bacterial strains to polymyxin B, suggesting a potential new approach to the treatment of MDR infections.

Objective: To evaluate the performance of whole-genome sequencing (WGS) for predicting Mycobacterium tuberculosis (MTB) drug resistance. Methods: 276 rifampin-resistance tuberculosis (RR-TB) and 30 rifampicin-sensitive clinical isolates were randomly selected from patients with tuberculosis in Shanghai Pulmonary Hospital (SPH). Phenotypic drug susceptibility testing (DST) against six anti-TB drugs was performed, and WGS was used to predict the drug resistance using an online ‘TB-Profiler’ tool. Results: Using phenotypic susceptibility as the gold standard, the overall sensitivities and specificities for WGS were 94.53% and 92.00% for isoniazid, 97.10% and 100.00% for rifampicin, 97.46% and 64.36% for ethambutol, 97.14% and 95.83% or streptomycin, 93.02% and 98.87% for ofloxacin, and 75.00% and 100.00% for amikacin, respectively. The concordances of WGS-based DST and phenotypic DST were: isoniazid (94.12%), rifampicin (97.39%), ethambutol (77.12%), streptomycin (96.73%), ofloxacin (96.41%) and amikacin (97.06%). Conclusions: WGS could be a promising approach to predict resistance to isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and amikacin.

Abstract Background Xpert MTB/RIF (Xpert) is an automated molecular test recommended by World Health Organization (WHO) for diagnosis of tuberculosis (TB). This study evaluated the effect of Xpert implementation on the detection of pulmonary TB (PTB) and rifampicin-resistant TB (RR-TB) cases in Shanghai, China. Methods Xpert was routinely implemented in 2018 for all presumptive PTB patients. All PTB patients above 15 years-old identified within the Provincial TB Control Program during the first half of each of 2017 and 2018, were enrolled to compare the difference in proportions of bacteriological confirmation, patients with drug susceptibility test (DST) results for rifampicin (ie, DST coverage) and RR-TB detection before and after Xpert’s implementation. Results A total of 6047 PTB patients were included in the analysis with 1691 tested by Xpert in 2018. Percentages of bacteriological confirmation, DST coverage and RR-TB detection in 2017 and 2018 were 50% vs. 59%, 36% vs. 49% and 2% vs. 3%, respectively (all p-values

ABSTRACT Mycobacterium kansasii, an important opportunistic pathogen of humans, causes serious pulmonary disease. Sixty M. kansasii isolates were collected for investigating the clinical characteristics of patients with M. kansasii infections as well as drug susceptibility and genotypes of M. kansasii. More than 90% of the patients infected with M. kansasii were from eastern China. According to the internal transcribed spacers (ITS), rpoB, hsp65, and tuf, all M. kansasii isolates were classified as molecular type I, irrespective of the disease manifestation. Sixty M. kansasii isolates from China were diverse and separated into four branches. Pairwise average nucleotide identity (ANI) values for M. kansasii isolates affiliated with different genotypes were more than 85%. The earliest isolate was isolated from Jiangsu in 1983. Of the isolates, 78.3% (47/60) were isolated since 1999. All isolates were sensitive to rifabutin. All but one isolate was sensitive to clarithromycin. Sensitivity rates to rifampin, amikacin, moxifloxacin, and linezolid were 80.0%, 90.0%, 88.3%, and 91.7%, respectively. A high rate of resistance was noted for ciprofloxacin (44 isolates, 73.3%) and ethambutol (46 isolates, 76.7%). Compared with M. tuberculosis H37Rv, 12 mutations of embCA were observed in all M. kansasii isolates. All these 60 M. kansasii isolates shared identical sequences of rpoB, inhA, katG, rrl, rrs, rpsL, gyrA, and gyrB. In conclusion, M. kansasii isolates are exhibiting greater genetic diversity globally. The resistance mechanism of M. kansasii is not necessarily related to gene mutation. IMPORTANCE M. kansasii type I is the main genotype spreading worldwide. The molecular history of the global spread of type I isolates remains largely unclear. We conducted a detailed analysis of genomic evolution of global M. kansasii isolates. Our results suggest that M. kansasii isolates exhibit greater genetic diversity globally.

The rise and global dissemination of extensively drug-resistant (XDR) bacteria are often related to plasmid-borne mobile antimicrobial resistance genes. Notably, isolates having multiple plasmids are often highly resistant to almost all the antibiotics available. In this study, we characterized an extensively drug-resistant Klebsiella pneumoniae 1678, which exhibited high-level resistance to almost all the available antibiotics. Through whole-genome sequencing (WGS), more than 20 resistant elements and 5 resistant plasmids were observed. Notably, the tigecycline resistance of K. pneumoniae 1678 was not related to the plasmid-borne tetA gene but associated with the overexpression of AcrAB and OqxAB efflux pumps, according to the susceptibility results of tetA-transformant and the related mRNA quantification of RND efflux pumps. Except for tigecycline resistance, three plasmids, mediating resistance to colistin, Fosfomycin, and ceftazidime–avibactam, respectively, were focused. Detailed comparative genetic analysis showed that all these plasmids belonged to dominated epidemic plasmids, and harbored completed conjugation systems. Results of conjugation assay indicated that these three plasmids not only could transfer to E. coli J53 with high conjugation frequencies, respectively, but also could co-transfer to E. coli J53 effectively, which was additionally confirmed by the S1-PFGE plasmids profile. Moreover, multiple insertion sequences (IS) and transposons (Tn) were also found surrounding the vital resistant genes, which may form several novel mechanisms involved in the resistant determinants’ mobilization. Overall, we characterized and reported the uncommon co-existence and co-transferring of FosA3-, NDM-5, and MCR-1-encoding plasmids in a K. pneumoniae isolate, which may increase the risk of spread of these resistant phenotypes and needing great concern.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) has augmented due to the abuse of antibiotics, bringing about difficulties in the treatment of infection especially with the formation of biofilm. Thus, it is essential to develop antimicrobials. Here we synthesized a novel small-molecule compound, which we termed SYG-180-2-2 (C21H16N2OSe), that had antibiofilm activity. The aim of this study was to demonstrate the antibiofilm effect of SYG-180-2-2 against clinical MRSA isolates at a subinhibitory concentration (4 μg/ml). In this study, it was showed that significant suppression in biofilm formation occurred with SYG-180-2-2 treatment, the inhibition ranged between 65.0 and 85.2%. Subsequently, confocal laser scanning microscopy and a bacterial biofilm metabolism activity assay further demonstrated that SYG-180-2-2 could suppress biofilm. Additionally, SYG-180-2-2 reduced bacterial adhesion and polysaccharide intercellular adhesin (PIA) production. It was found that the expression of icaA and other biofilm-related genes were downregulated as evaluated by RT-qPCR. At the same time, icaR and codY were upregulated when biofilms were treated with SYG-180-2-2. Based on the above results, we speculate that SYG-180-2-2 inhibits the formation of biofilm by affecting cell adhesion and the expression of genes related to PIA production. Above all, SYG-180-2-2 had no toxic effects on human normal alveolar epithelial cells BEAS-2B. Collectively, the small-molecule compound SYG-180-2-2 is a safe and effective antibacterial agent for inhibiting MRSA biofilm.

The significant increase in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to fusidic acid (FA) is a worrying public concern. However, the data on the prevalence of FA-resistant MRSA isolates in China is still limited. This study aims to investigate the prevalence of FA resistance and resistance determinants among MRSA isolates from six tertiary hospitals in different regions of China between 2016 and 2020. The antimicrobial susceptibility of MRSA isolates was performed by disk diffusion test and broth microdilution method. Whole-genome sequencing was conducted to evaluate the determinants of FA resistance and molecular characterization of FA-resistant MRSA isolates. In this study, a total of 74 (74/457, 16.2%) isolates were identified to be FA-resistant among 457 non-duplicate MRSA isolates. The prevalence of 74 FA-resistant isolates was as follows: Hubei (28/70, 40%), Shanghai (18/84, 21.4%), Jiangxi (7/58, 12.1%), Inner Mongolia Autonomous Region (6/38, 15.8%), Guangdong (12/112, 10.7%), and Sichuan (3/95, 3.2%). The mutations in fusA were present in 79.7% (59/74) of FA-resistant MRSA isolates, with 54 (54/74, 73%) having L461K mutation and conferring high-level resistance [Minimum Inhibitory Concentration (MIC)>128 μg/ml]. Acquired gene, fusB, with low-level resistance (MIC

Urinary tract infection (UTI) is one of the most common bacterial infections and UTI is the most common extraintestinal infectious disease entity in women worldwide. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTI. While antimicrobial resistance has emerged as one of the principal problems of UTI, little is known about the epidemiology of UPEC isolated from female patients in Shanghai. This study aimed to describe the antimicrobial resistance and molecular epidemiology of UPEC isolated from female patients in Shanghai, China. UPEC isolates were collected from female patients from July 2019 to June 2020 in Shanghai and a total of 151 isolates were obtained randomly. Antimicrobial susceptibility testing was performed using the disk diffusion method. Multilocus sequencing type, phylogenetic groups, antimicrobial resistance genes, and virulence genes were detected by polymerase chain reaction. In our study, no carbapenem-resistant isolates were found, but fluoroquinolone-resistant and multi-drug resistant UPEC accounted for 62.25% and 42.38%, respectively. The phylogenetic group B2 (58.94%) predominated, followed by phylogenetic group D (26.49%). The most prevalent sequence type was ST1193 (25.83%), which was first reported in Shanghai. The rate of extended-spectrum β-lactamase (ESBL)-positive isolates was 39.74% and the dominant ESBL genotype was blaCTX-M-14 (21/60), followed by blaCTX-M-55 (12/60). Mutations in gyrA were detected in the majority of fluoroquinolone-resistant isolates (90/94), followed by parC (85/94) and parE (71/94). The aac (3) -IIa was also found in 85% of aminoglycoside resistance isolates. Among 151 UPEC isolates, the common virulence genes were csgA (97.35%), fimH (92.72%), sitA (82.12%), and malX (65.56%). In conclusion, the high antimicrobial resistance of UPEC isolated from female patients, harboring a series of virulence genes, are troublesome for medical practitioners in Shanghai. At present, the prevalent ST1193 and emerging blaCTX-M-55 make UTI therapy more challenging.

Abstract Background Staphylococcus epidermidis has emerged as an often encountered pathogen responsible for hospital-acquired infections. The aim of present study is to investigate the microbiological characteristic of S. epidermidis isolates isolated from sterile specimens and skin in a Chinese tertiary hospital. Methods A total of 223 non-duplicate S. epidermidis were collected from various sterile specimens of inpatients among 10 years in Wenzhou, China. 106 S. epidermidis obtained from the skin (urethral orifices) of healthy volunteers. All isolates were tested for antimicrobial susceptibility. PCR was used to detect the virulence- and resistance-associated genes and 7 housekeeping genes to determine the sequence types (STs) of selected isolates. Results The resistance rates to antimicrobials tested except linezolid and vancomycin and the prevalence of methicillin-resistant S. epidermidis (MRSE) of S. epidermidis clinical isolates were significantly higher than those among colonized isolates (P 90%), which was similar to the characteristics of ST59 clone with one locus difference from ST466. ST466 clone competence with Staphylococcus aureus was relatively stronger, relative to ST2, ST20, ST130 and ST59 clones. Conclusion Taken together, a high-level of genetic diversity was found between clinical and colonized S. epidermidis isolates. A novel ST466 clone with distinct and similar characteristics relative to other prevalent clones, emerging as a prevalent clone in China, should be of major concern.

Objective: To evaluate the diagnostic performance of Xpert MTB/RIF Ultra for EPTB (Extrapulmonary Tuberculosis) patients on different types of extrapulmonary specimens from different anatomic sites. Methods: Patients with suspected EPTB were prospectively included, extrapulmonary specimens were collected and subjected to culture, Xpert and Xpert Ultra assays in accordance with relevant guidelines. Results: A total of 225 cases were included which contained 200 EPTB cases (43 culture-positive EPTB, 157 culture-negative EPTB which were diagnosed based on pathological results and a satisfied response to anti-TB treatment) and 25 non-EPTB cases. Sensitivities of Xpert Ultra and Xpert for culture-positive cases were 83.7% (95%CI, 68.7–92.7) and 67.4% (95% CI, 51.3–80.5) respectively. Specificities of Xpert Ultra and Xpert were 92.0% (95% CI, 72.5–98.6) and 96.0% (95% CI, 77.7–99.8) respectively. The sensitivities of Xpert Ultra, Xpert and culture for 200 EPTB cases were 52.5% (105/200, 95% CI, 45.4–59.6), 34.0% (68/200, 95% CI, 27.6–41.1) and 21.5% (43/200, 95% CI, 16.2–28.0) respectively. By comparison among different types of specimens, Xpert Ultra can detect 78.9% (56/71) of EPTB on fine-needle aspiration (FNA) tissues which was higher than that on pleural fluid (43.7% (45/103), p