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Fengjun Xia,1,2,* Xiaoyan Tao,2,3,* Haichen Wang,1,2 Jian Shui,4 Changhang Min,1,2 Yubing Xia,1,2 Jun Li,1,2 Mengli Tang,1,2 ZhaoJun Liu,1,2 Yongmei Hu,1,2 Huidan Luo,1,2 Mingxiang Zou1,2 1National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 2Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 3Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China; 4Department of Clinical Laboratory, Changsha Central Hospital, Changsha, 410008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Mingxiang Zou, National Clinical Research Center for Geriatric Disorders, Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, Hunan Province, 410008, People’s Republic of China, Tel/Fax +86-7384327440, Email zoumingxiang@csu.edu.cnPurpose: As an under-explored biomaterial, bacterial biofilms have a wide range of applications in the green synthesis of nanomaterials. The biofilm supernatant of Pseudomonas aeruginosa PA75 was used to synthesize novel silver nanoparticles (AgNPs). BF75-AgNPs were found to possess several biological properties.Methods: In this study, we biosynthesized BF75-AgNPs using biofilm supernatant as the reducing agent, stabilizer, and dispersant and investigated their biopotential in terms of antibacterial, antibiofilm, and antitumor activities.Results: The synthesized BF75-AgNPs demonstrated a typical face-centered cubic crystal structure; they were well dispersed; and they were spherical with a size of 13.899 ± 4.036 nm. The average zeta potential of the BF75-AgNPs was − 31.0 ± 8.1 mV. The BF75-AgNPs exhibited strong antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase Escherichia coli (ESBL-EC), extensively drug-resistant Klebsiella pneumoniae (XDR-KP), and carbapenem-resistant Pseudomonas aeruginosa (CR-PA). Moreover, the BF75-AgNPs had a strong bactericidal effect on XDR-KP at 1/2 × MIC, and the expression level of reactive oxygen species (ROS) in bacteria was significantly increased. A synergistic effect was observed when the BF75-AgNPs and colistin were used for the co-treatment of two colistin-resistant XDR-KP strains, with fractional inhibitory concentration index (FICI) values of 0.281 and 0.187, respectively. Furthermore, the BF75-AgNPs demonstrated a strong biofilm inhibition activity and mature biofilm bactericidal activity against XDR-KP. The BF75-AgNPs also exhibited a strong antitumor activity against melanoma cells and low cytotoxicity against normal epidermal cells. In addition, the BF75-AgNPs increased the proportion of apoptotic cells in two melanoma cell lines, and the proportion of late apoptotic cells increased with BF75-AgNP concentration.Conclusion: This study suggests that BF75-AgNPs synthesized from biofilm supernatant have broad prospects for antibacterial, antibiofilm, and antitumor applications.Graphical Abstract: Keywords: multidrug resistant, green synthesis, colistin, metallic silver, melanoma

Background The prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak. Results The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored blaKPC-2, blaNDM-1, and the virulence-related genes prmpA, prmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8–20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying blaKPC-2, blaNDM-1, blaSHV-12, blaLAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance. Conclusions Polymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Haichen Wang,1,2 Jia Li,2,3 Changhang Min,1,2 Fengjun Xia,1,2 Mengli Tang,1,2 Jun Li,1,2 Yongmei Hu,1,2 Mingxiang Zou1,2 1National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan Province, People’s Republic of China; 2Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, Hunan Province, People’s Republic of China; 3Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of ChinaCorrespondence: Mingxiang Zou, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, 41008, People’s Republic of China, Tel/Fax +86 7384327440, Email zoumingxiang@csu.edu.cnPurpose: Due to the extensive consumption of silver-containing compound, silver resistance spreads among gram-negative pathogens and is regarded as a great public problem. In this study, we investigated silver resistance mechanisms and antibiotic resistance genes co-harbored with sil operon among gram-negative pathogens isolated from wound samples.Methods: A total of 193 strains of gram-negative pathogens were collected from wound samples between 2018 and 2020 in Xiangya hospital. Silver resistance was obtained by broth microdilution method. The silver resistance mechanisms and the prevalence, genetic environments, and coexistence with antibiotic resistance genes of sil operon were investigated by polymerase chain reaction (PCR) and whole genome sequencing (WGS).Results: Among 193 strains, nine strains (4.7%) were resistant to Ag+ and assigned to the following species: Klebsiella pneumoniae (n = 5) and Enterobacter hormaechei (n = 4). WGS confirmed that 24 strains carried the entire sil operon, including the four Ag+-resistant E. hormaechei and 20 Ag+-susceptible strains, while PCR failed to detect some sil genes, especially silE, due to sequence variations. In seven strains, Tn7 transposon was identified in the upstream of sil operon. Spontaneous mutants resistant to Ag+ were induced in 15 out of 20 Ag+-susceptible strains, including K. pneumoniae strains belonged to high-risk groups (ST11 and ST15). The sil-positive strains harbored various antibiotic resistance genes, including blaESBL and blaApmC. WGS revealed that a single mutation in cusS gene and loss of major porins conferred silver resistance in the five K. pneumoniae strains.Conclusion: Our findings emphasize the cryptic silver resistance is prevalent among Enterobacteriaceae with sil operon or with the combination of cus operon and major porin loss and increase the understanding of the prevalence of sil operon with antibiotic resistance genes, especially blaESBL and blaApmC.Keywords: silver resistance, gram negative pathogen, sil operon, antibiotic resistance gene, whole-genome sequencing

Introduction Cryptococcemia is associated with poor prognosis among patients with cryptococcosis. However, there are limited data on the clinical features of cryptococcemia, particularly among patients with different immune statuses. This study assessed the largest number of cases diagnosed with cryptococcemia, to the best of our knowledge. Methods Demographic and clinical data of patients with positive blood culture results for Cryptococcus were obtained from medical records at the Xiangya Hospital (2010–2019). Results A total of 65 patients were diagnosed and treated for cryptococcemia, of which 53 (82%) immunosuppressed patients were afflicted with HIV (12%, 8/65), tuberculosis (8%, 5/26), liver cirrhosis (6%, 3/65), chronic renal failure (6%, 3/65), nephrotic syndrome (13%, 7/65), systemic lupus erythematosus (8%, 5/65), chronic glomerulonephritis (11%, 6/65), malignant diseases (19%, 10/65), and diabetes (11%, 6/65). Most patients (85%, 55/65) presented with fever. Other symptoms, such as cough, headache, enlarged lymph nodes, liver, or spleen, and septic shock, were also reported. Typically, the sites of infection included the central nervous system, lung, skin, bone, abdomen, endometrium, lymph node, and blood. Although early systemic antifungal therapy was administered to 61 patients within 48 h of hospitalization, the 60-day mortality rate was higher in the immunosuppressed group (53%) than in the immunocompetent group (8%). Conclusions Our study indicated that patients with different immune statuses presented different clinical features. Immunosuppressed patients with cryptococcemia presented a higher risk of mortality with poor prognosis, which required intense attention and treatment in time.

OBJECTIVE: To investigate the relationship of biofilm-forming ability of Pseudomonas aeruginosa(PA) with swimming motility, twitching motility and virulence gene distribution. METHODS: A total of 192 clinical isolates of PA were collected consecutively. Microtiter plate method was used to evaluate the ability to form biofilm. The swimming and twitching motilities were detected by plate method. Polymerase chain reaction (PCR) was used to detect virulence genes. RESULTS: Of the 192 PA clinical isolates, 186 (96.9%) showed biofilm-forming ability. Among them, 36 isolates showed weak biofilm-forming ability, 84 exhibited moderate biofilm-forming ability and 66 showed strong biofilm-forming ability. The diameters of the swimming ring for PA with none biofilm-forming ability, weak biofilm-forming ability, moderate biofilm-forming ability, strong biofilm-forming ability were (9.12±6.76), (18.42±7.51), (19.10±4.77) and (17.80±5.27) mm, respectively. The diameters of the twitching ring for PA in above groups were (8.38±1.50), (17.21±7.42), (18.49±5.62) and (20.44±6.43) mm, respectively. The swimming motility and twitching motility of none biofilm-forming ability group were weaker than biofilm-forming ability groups (all P

Background Carbapenem-resistant Enterobacterales (CRE) are rapidly increasing worldwide in last two decades and lead few antibiotics for treatment. The molecular epidemiology of CRE in China was investigated to provide basis for clinical rational use of antibiotics and prevent its spread. Methods All CRE isolates in this study were collected from 11 hospitals from October 2015 to July 2018. The isolates were subjected to antimicrobial susceptibility tests, PCR molecular identification, pulsed-field gel electrophoresis, and multilocus sequence typing. Results Among the 399 CRE isolates, 51.6% (206/399) harbored carbapenemase genes. Three carbapenemase genes were detected, namely blaKPC-2, blaNDM-1, and blaIMP at rates of 29.8% (119/399), 17.5% (70/399), and 4.0% (16/399), respectively. In Klebsiella pneumoniae (350) and Escherichia coli (26), blaKPC-2 (33.4%, 117/350) and blaNDM-1 (61.5%, 16/26) were the predominant genes. The most common genes in the CRE isolates were blaKPC (85.5%) and blaNDM-1 (76.5%) from adults and children, respectively. Particularly, ST11 K. pneumoniae with blaKPC-2 harbored by IncFII plasmids were distributed in both general and primary hospitals, suggesting a clonal transmission pattern at these sites. In addition, the clonal distribution of ST2407 K. pneumoniae with blaNDM-1 located on IncX3 plasmids and blaIMP-38-positive ST307 K. pneumoniae were detected in a children’s hospital. Conclusion The distribution of carbapenemase genes differed among strains and age groups. Multiple carbapenemase genes in the CRE strains were clonally disseminated in the tested regions mediated by multiple plasmids. Therefore, CRE monitoring should be increased and measures should be adopted to prevent its transmission.

Background Early and accurate diagnosis of microorganism(s) is important to optimize antimicrobial therapy. Shotgun metagenomic sequencing technology, an unbiased and comprehensive method for pathogen identification, seems to potentially assist or even replace conventional microbiological methodology in the diagnosis of infectious diseases. However, evidence in clinical application of this platform is relatively limited. Methods To evaluate the capability of shotgun metagenomic sequencing technology in clinical practice, both shotgun metagenomic sequencing and conventional culture were performed in the PCR-positive body fluid specimens of 20 patients with suspected infection. The sequenced data were then analyzed for taxonomic identification of microbes and antibiotic resistance gene prediction using bioinformatics pipeline. Results Shotgun metagenomic sequencing results showed a concordance of 17/20 compared with culture results in bacterial detection, and a concordance of 20/20 compared with culture results in fungal detection. Besides, drug-resistant types annotated from antibiotic resistance genes showed much similarity with antibiotic classes identified by susceptibility tests, and more than half of the specimens had consistent drug types between shotgun metagenomic sequencing and culture results. Conclusions Pathogen identification and antibiotic resistance gene prediction by shotgun metagenomic sequencing identification had the potential to diagnose microorganisms in infectious diseases, and it was especially helpful for multiple microbial co-infections and for the cases where standard culture approached failed to identify microorganisms.

Additional file 1: Figure S1. PCR analysis of genes blaKPC-2 (A) and rmtB (B) of fosA3 transconjugants. M = Marker. 1, E. coli J53 (KP5 plasmid); 2, E. coli J53 (KP 18 plasmid); 3, E. coli J53 (KP 165 plasmid); 4, E. coli J53 (KP 190 plasmid); 5, E. coli J53 (KP 212 plasmid); 6, E. coli J53 (KP 223 plasmid).

Cryptococcal meningitis, mainly caused by Cryptococcus neoformans/gattii species complexes, is a lethal infection in both immunosuppressive and immunocompetent populations. We characterized 110 Cryptococcus strains collected from Xiangya Hospital of Central South University in China during the 6-year study period between 2013 and 2018, and performed their antifungal susceptibility testing. Furthermore, the clinical features, laboratory and imaging data, treatment strategies and outcomes of the subjects were retrospectively analyzed. Of 110 Cryptococcus strains, C. neoformans species complexes accounted for 96.4% (106/110), including C. neoformans sensu stricto (VNI molecular type, 95.5%, 105/110) and Cryptococcus deneoformans (VNIV molecular type, 0.9%, 1/110), and Cryptococcus deuterogattii (VGII molecular type) accounted for 3.6% (4/110). The strains were further classified into 17 individual sequence types (STs) by using multilocus sequence typing (MLST). 89.1% (98/110) were represented by ST5; seven C. deuterogattii strains and one Cryptococcus deneoformans strain were assigned as ST7 and ST260, respectively. Antifungal minimal inhibitory concentrations above the epidemiological cutoff values (ECVs) were found mainly in C. neoformans species complexes strains (nine for amphotericin B, nine for fluconazole and seven for 5-fluorocytosine). Furthermore, 60.9% (67/110) of the subjects were male, and 40.0% (44/110) did not have underlying diseases. Hepatic diseases (hepatitis/HBV carrier status and cirrhosis) were the most common underlying health conditions (11.8%, 13/110), followed by autoimmune disorders (10.9%, 12/110) and chronic kidney disease (6.36%, 7/110). Only 4.5% (5/110) of the patients were HIV/AIDS positives. For clinical presentation, headache (77.3%, 85/110), fever (47.3%, 52/110), and stiff neck (40.9%, 45/110) were commonly observed. The mortality rate was 35.0% (36/103). In conclusion, our data were characterized by a high prevalence of the Cryptococcal meningitis patients without HIV/AIDS and other underlying health conditions, a relatively high non-wild-type rate of fluconazole and amphotericin B resistance, and low genetic diversity in Cryptococcus strains. The present study will provide evidence for further improvement of the diagnosis and treatment of cryptococcosis in China.

Background: Cryptococcemia is associated with poor prognosis among patients with cryptococcal diseases; however, limited data are available on the clinical features of cryptocoaemia. Methods: A retrospective cohort study was conducted to describe the clinical features of cryptococcaemia. Demographic and clinical data of patients with positive blood culture of cryptococcus were obtained from the Xiangya hospital (2010–2019). Demographic and clinical information was obtained from medical records. Results: Sixty-five patients were diagnosed and treated for cryptococcaemia out of which 53 (82%, 53/65) immunosuppressed patients were afflicted with HIV (12%, 8/65), tuberculosis (8%, 5/26), liver cirrhosis (6%, 3/65), chronic renal failure (6%, 3/65), nephrotic syndrome (13%, 7/65), systemic lupus erythematosus (8%, 5/65, chronic glomerulonephritis (11%, 6/65), malignant diseases (19%, 10/65), and Diabetes (11%, 6/65). Most patients (85%, 55/65), presented with fever. Other symptoms such as cough, headache, enlarged lymph nodes, liver or spleen, and septic shock were also reported. Typically, the sites of infection included the central nervous system, lung, skin, bone, abdomen, endometrium, lymph node, and blood. Although an early systemic antifungal therapy was given to sixty-one patients within 48h of hospitalization, the 60-day mortality was higher in immunosuppressed group (53%) than the immunocompetent group (8%). Conclusions: Our study indicated that fever, sepsis, and meningeal involvement were the most common clinical features of cryptococcaemia. Receiving treatment with immunosuppressive drugs, malignant diseases and HIV infection were the three major predisposing conditions. The immunosuppressed patients with cryptococcaemia presented a higher risk of mortality with poor prognosis.

Following the discovery and emergence of the plasmid-mediated colistin resistance gene, mcr-1, the Chinese government formally banned colistin as an animal growth promoter on April 30, 2017. Herein, we report patterns in colistin resistance and mcr-1 abundance in Escherichia coli from animals and humans between 2015 and 2019, to evaluate the effects of the colistin withdrawal.We did an epidemiology comparative study to investigate: annual production and sales of colistin in agriculture across mainland China according to data from the China Veterinary Drug Association from 2015 to 2018; the prevalence of colistin-resistant E coli (CREC) in pigs and chickens in 23 Chinese provinces and municipalities as reported in the China Surveillance on Antimicrobial Resistance of Animal Origin database from Jan 1, 2015, to Dec 31, 2016, and Jan 1, 2017, to Dec 31, 2018; the presence of residual colistin and mcr-1 in faeces from 118 animal farms (60 pig, 29 chicken, and 29 cattle) across four provinces over July 1, 2017, to August 31, 2017, and July 1, 2018 to August 31, 2018; the prevalence of mcr-1-positive E coli (MCRPEC) carriage in healthy individuals attending routine hospital examinations across 24 provinces and municipalities from June 1 to July 30, 2019, comparing with equivalent 2016 data (June 1 to September 30) from our previous study in the same hospitals; and the patterns in CREC prevalence among hospital E coli infections across 26 provinces and municipalities from Jan 1, 2015, to Dec 31, 2016, and Jan 1, 2018, to Dec 31, 2019, reported on the China Antimicrobial Surveillance Network.After the ban on colistin as a growth promoter, marked reductions were observed in the production (27 170 tonnes in 2015 vs 2497 tonnes in 2018) and sale (US$71·5 million in 2015 vs US$8·0 million in 2018) of colistin sulfate premix. Across 118 farms in four provinces, mean colistin residue concentration was 191·1 μg/kg (SD 934·1) in 2017 versus 7·5 μg/kg (50·0) in 2018 (p0·0001), and the median relative abundance of mcr-1 per 16S RNA was 0·0009 [IQR 0·0001-0·0059] in 2017 versus 0·0002 [0·0000-0·0020] in 2018 (p=0·0001). Across 23 provinces and municipalities, CREC was identified in pig faeces in 1153 (34·0%) of 3396 samples in 2015-16 versus 142 (5·1%) of 2781 in 2017-18 (p0·0001); and in chickens in 474 (18·1%) of 2614 samples in 2015-16 versus 143 (5·0%) of 2887 in 2017-18 (p0·0001). In hospitals across 24 provincial capital cities and municipalities, human carriage of MCRPEC was identified in 644 (14·3%) of 4498 samples in 2016 versus 357 (6·3%) of 5657 in 2019 (p0·0001). Clinical CREC infections in 26 provinces and municipalities comprised 1059 (1·7%) of 62 737 E coli infections in 2015-16 versus 794 (1·3%) of 59 385 in 2018-19 (p0·0001).The colistin withdrawal policy and the decreasing use of colistin in agriculture have had a significant effect on reducing colistin resistance in both animals and humans in China. However, continuous colistin monitoring is essential, in particular to act as an early warning system for colistin stewardship in Chinese hospitals.National Key Research and Development Program of China, National Natural Science Foundation of China, and UK Medical Research Council.

Carbapenem-resistant K. pneumoniae (CR-KP) posts significant public health challenge worldwide. The aim of this study is to assess clinical characteristics and molecular epidemiology of CR-KP infections with Multilocus sequence typing (MLST) and Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) in Central China. A total of 71 CR-KP isolates were recovered in a teaching hospital from October 2014 to December 2015. Among all CR-KP isolates, 73.2% (52) produced K. pneumoniae carbapenemases-2 (KPC-2). Eighteen ST types were identified by MLST, among these ST types, forty-seven isolates belonged to ST11 type, which was the predominant outbreak strain in China, and most ST11 isolates produced KPC-2. Eleven mass spectrometry (MS) types were identified by MALDI-TOF MS analysis, 53.5% isolates were MS4 and MS6, which matched with ST11 in MLST analysis. CR-KP infection was associated with increased medical cost and longer hospitalization. Therefore, we found that KPC-2-producing ST11 (MS4 and MS6) CR-KP isolates were the predominant clone identified by MLST and MALDI-TOF, and CR-KP infection was associated with increased hospital costs and longer hospitalization.

OBJECTIVE: To investigate the phylogenetics and prevalence of bloodstream infections with Escherichia coli ST131, the antimicrobial resistance profiles of the pathogens, and the clinical features. METHODS: Non-duplicate Escherichia coli isolates were collected from 144 patients with bloodstream infections in our hospital between January and December, 2016.The phylogenetic groups of the isolates were analyzed using multiplex PCR, and O serotyping of ST131 strains was performed by allele-specific PCR.The clinical characteristics of the 144 patients were analyzed to define the differences in the clinical features between patients with ST131 infection and those with non-ST131 infection.Antibiotic susceptibility of the isolates was determined using the Vitek 2 compact system. RESULTS: The phylogenetic group analysis showed a domination by group B2 (41.0%[59/144]), followed by group F, group B1 and group E, which accounted for 16.7%(24/144), 13.9%(20/144), and 13.2% (19/144), respectively.Nine strains (6.3%) of Escherichia coli were identified to be ST131 strains, among which 8 were O25b-B2-ST131 strains and 1 was O16-B2-ST131 strain.Of the 9 cases of ST131 infection, 7(77.8%) were found to occur in a nosocomial setting.The demographic characteristics and clinical features of the ST131-infected patients were similar to those of non-ST131-infected patients.ST131 strains were sensitive to piperacillin/tazobactam, imipenem, ertapenem, and amikacin, but showed high resistance rates to cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, gentamicin, and trimethoprim/ sulfamethoxazole (all over 50%).The positivity rate of ESBLs in the ST131 strains was 77.8%, and the multidrug resistance rate reached 88.9%, which was higher than that of non-ST131 isolates, but the difference was not statistically significant. CONCLUSIONS: The most common phylogenetic groups of Escherichia coli isolates from patients with bloodstream infections are group B2 and F, and the positivity rate of ST131 is low.We for the first time detected O16-ST131 in patients with blood-borne infections in China.The clinical features of ST131-infected patients are similar to those of non-ST131-infected patients.The positivity rate of ESBLs and the multidrug resistance rate are high in ST131 strains, which may raise concerns in the future.

The purpose of this study was to investigate the clinical characteristics of hypervirulent K. pneumoniae (hvKP) induced ventilator-associated pneumonia (VAP) and the microbiological characteristics and epidemiology of the hvKP strains. A retrospective study of 49 mechanically ventilated patients with K. pneumoniae induced VAP was conducted at a university hospital in China from January 2014 to December 2014. Clinical characteristics and K. pneumoniae antimicrobial susceptibility and biofilm formation were analyzed. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence factors plasmid rmpA(p-rmpA), iroB, iucA, mrkD, entB, iutA, ybtS, kfu and allS were also evaluated. Multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) analyses were used to study the clonal relationship of the K. pneumoniae strains. Strains possessed p-rmpA and iroB and iucA were defined as hvKP. Of 49 patients, 14 patients (28.6 %) were infected by hvKP. Antimicrobial resistant rate was significantly higher in cKP than that in hvKP. One ST29 K54 extended-spectrum-beta-lactamase (ESBL) producing hvKP strain was detected. The prevalence of K1 and K2 in hvKP was 42.9 % and 21.4 %, respectively. The incidences of K1, K2, K20, p-rmpA, iroB, iucA, iutA, Kfu and alls were significantly higher in hvKP than those in cKP. ST23 was dominant among hvKP strains, and all the ST23 strains had identical RAPD pattern. hvKP has become a common pathogen of VAP in mechanically ventilated patients in China. Clinicians should increase awareness of hvKP induced VAP and enhance epidemiologic surveillance.

Intervertebral disc (IVD) degeneration (IDD) is a major contributor to low back pain. During IDD progression, ROS can be produced in the form of H

BackgroundStaphylococcus aureus is a leading cause of nosocomial infections. The purpose of this study was to evaluate the prevalence of methicillin-resistant S. aureus (MRSA) and heterogeneous vancomycin-intermediate S. aureus (hVISA), and compare the antimicrobial susceptibility, molecular characteristic, and virulence factors in methicillin-susceptible S. aureus (MSSA), MRSA, and hVISA from central-southern China.MethodsA total of 184 S. aureus were isolated from sterile body fluids. All isolates were subjected to population analysis profiling for the identification of hVISA phenotype and polymerase chain reaction analysis for genotyping and 31 virulence genes.ResultsThe prevalence of MRSA isolates was 41.8% in central-southern China. Of 77 MRSA isolates, 17 (22.1%) were identified as hVISA. The most common MRSA and MSSA clones were ST239-MRSA-SCCmecIII-t030-agr-I (55.8%) and ST188-MSSA-t189-agr-I (20.6%), respectively. The frequency of carriage of pvl, hemolysins, tst, and staphylococcal enterotoxin genes among MSSA isolates was significantly higher than that for MRSA isolates (p

A simplified Carba NP test (CNPt), the CNPt-direct, was evaluated for carbapenemase detection using 272 gram-negative bacteria. Compared to the Modified Hodge test, the CNPt-direct had higher sensitivity (98.2% vs. 71.6%). The simplified Carba NP is an accurate, cheap and simple test for rapid carbapenemase detection.

To investigate relationship between AdeABC efflux pump and resistance of Acinetobacter baumannii against carbapenem. Methods: Carbapenem-resistant strains were acquired from multistep selection resistance test by meropenem in vitro. The quantitation test for sensitivities of strains before and after induction was determined by the E-test, and carbonylcyanide-m-chlorophenylhydrazone (CCCP) inhibition test was used to screen efflux pump. PCR, sequencing analysis, or real-time PCR was used to analyze the changes of regulatory genes adeR and adeS of the AdeABC efflux pump system, or expressions of adeA, adeB, adeR, and adeS in the strains before and after induction, respectively. Results: The minimal inhibitory concentrations (MICs) of meropenem were at 0.38 μg/mL and 0.25 μg/mL in parental sensitive strain S25595 and S7257, respectively, and the MICs of meropenem for both S25595 and S7257 after induction were more than 32 μg/mL. Compared with parental sensitive strains, the expression level of adeA, adeB, adeR, and adeS mRNA were elevated from 2.45 to 9.44 times, but there were no gene mutations or insertion sequences in the regulatory gene adeS and adeR. Conclusion: High expression of the AdeABC efflux pump system in Acinetobacter baumannii is closely associated with meropenem resistance. The upregulation of adeA and adeB expression is not due to gene mutations in the regulatory gene adeS and adeR and other mechanisms might account for it.目的：探讨外排泵AdeABC系统与鲍曼不动杆菌对碳青霉烯类药物耐药的关系。方法：采用美罗培南多步法体外诱导敏感鲍曼不动杆菌获得对碳青霉烯类药物耐药的菌株；采用E-test法定量检测诱导前后菌株的敏感性；羰基氰化物间氯苯腙(carbonylcyanide-m-chlorophenylhydrazone，CCCP)抑制试验筛查外排泵；PCR及测序分析诱导前后AdeABC系统的调控基因adeS，adeR及主要碳青霉烯酶基因的变化；荧光定量PCR检测诱导前后adeA，adeB，adeR和adeS基因mRNA的表达量。结果：亲代敏感菌株S25595和S7257的美罗培南最低抑菌浓度(minimal inhibitory concentration，MIC)分别为0.38和0.25 μg/mL，诱导后MIC均32 μg/mL；与亲代敏感株相比，诱导耐药株adeA，adeB，adeR和adeS基因的mRNA表达量上升2.45~9.44倍，但调控基因adeS和adeR没有基因突变或插入序列。结论：外排泵AdeABC系统高表达与鲍曼不动杆菌对美罗培南耐药密切相关，其表达水平升高不是由调控基因adeS和adeR序列中基因突变或插入序列引起，可能存在其他机制。.

The objectives of this study were to determine species distribution and in vitro antifungal susceptibility of Candida isolates identified in the multicentre China-SCAN study of invasive Candida infection (ICI) in intensive care units (ICUs) across China.Candida isolates from patients in the China-SCAN study with documented ICI were evaluated by a central laboratory. Species were identified using chromogenic culture media or the API 20C AUX kit. Susceptibility to fluconazole, voriconazole, itraconazole, caspofungin and amphotericin B was determined using the CLSI broth microdilution method (M27-A3) and updated clinical breakpoints or epidemiological cut-off values.A total of 389 isolates from 244 patients were analysed. Species identified most frequently were Candida albicans (40.1%), Candida parapsilosis (21.3%), Candida tropicalis (17.2%) and Candida glabrata (12.9%). Rarer species such as Lodderomyces elongisporus and Candida ernobii were also identified. Fluconazole susceptibility was evident in 85.9% (134/156) of C. albicans, 62.7% (42/67) of C. tropicalis and 48.2% (40/83) of C. parapsilosis isolates. Susceptibility to voriconazole was ≥ 90% among all species. All isolates were susceptible to amphotericin B and caspofungin except C. glabrata [86.0% (43/50) susceptible to caspofungin]. Cross-resistance between fluconazole and voriconazole was observed for C. parapsilosis and C. glabrata.Although C. albicans was the predominant single species, non-albicans species constituted50% of isolates. Fluconazole susceptibility was lower in most non-albicans species, indicating that fluconazole resistance should be closely monitored. Susceptibility to voriconazole, amphotericin B and caspofungin is encouraging. Differences between these data and those from other regions emphasize the importance of assessing regional variations.

Carbapenem-resistant Acinetobacter baumannii has been known as an opportunistic pathogen that causes a variety of illness worldwide. In this study, we characterize the molecular epidemiology of 174 non-repetitive clinical isolates of A. baumannii collected from January to June 2009 from Xiangya Hospital, in Hunan Province, China, including an outbreak period of A. baumannii. These 174 isolates harbored A. baumannii intrinsic gene OXA-51. They were resistant to multiple antibiotics with resistance rates as 49.4% to imipenem, 48.3% to meropenem, 46.6% to ampicillin-sulbactam, 6.9% to cefoperazone-sulbactam, and 6.3% to minocycline. 74 out of 174 isolates were identified as carbapenemase-producing strains, among which bla(OXA-23) gene was found in 71 isolates. These 74 carbapenemase expression strains could be divided into four genotypes by enterobacterial repetitive intergenic consensus (ERIC)-PCR, with 19, 17, 33 and 5 clones in each group. We also found four imipenem resistant isolates carrying OXA-23 gene without showing carbapenemase phenotype. Our findings show that the bla(OXA-23) gene is the common carbapenemase gene among carbapenem-resistant Acinetobacter spp. isolates, suggesting that clonal spread of carbapenemase-producing isolates may be one important factor which results in the high carbapenem resistance rate in the local hospital in Hunan Province, China.

To investigate the spectrum and antimicrobial resistance of major pathogensthat causing nosocomial infections in China, 2013.Nosocomial cases as well as pathogens causing bloodstream infections (BSI), hospital-acquired pneumonia (HAP) and intra-abdominal infections (IAI) from 13 teaching hospital around China were collected. The minimum inhibitory concentrations (MICs) were determined by the agar dilution method. The CLSI M100-S23 criteria were used for interpretation.Of all cases, 1 022 cases were from BSI, 683 from HAP and 674 from IAI.Escherichia coli and Klebsiella pneumoniae were the most prevalent pathogens causing BSI and IAI while Acinetobacter baumanii (34.6%) and Pseudomonas aeruginosa were dominated in HAP. Tigecycline, imipenem and meropenem exhibited high potency against Enterobacteriaceae and the susceptibilities rates were 95.6%, 94.2%and 95.2% respectively. Enterobacteriaceae demonstrated high resistance against cephalosporins (52.3%) and fluoroquinolones (38.9%) but were susceptible to β-lactam+inhibitor. Of all the Enterobacteriaceae, 30.5% were ESBLs positive and 4.3% were carbapenem resistant. Acinetobacter baumanii showed low susceptibilities to the microbial agents except for tigecycline (90.5%) and colistin (100%). The rate of carbapenem resistant Acinetobacter baumanii was 76.6%. Amikacin, ciprofloxacin, cefepime and piperacillin/tazobactam showed high antibacterial activity against Pseudomonas aeruginosa with susceptible rate 88.5%, 77.6%, 72.7% and 64.5% respectively. The resistant rate to imipenem and meropenem were 42.1% and 32.2%. All Staphylococcus aureus (166 strains) were susceptible to tigecycline, linezolid, daptomycin and glycopeptides. MRSA accounted for 46.9% of all the Staphylococcus aureus. The prevalence of MRSA in IAI (55.2%) and HAP (54.4%) were higher that that in BSI (35.0%). No Enterococcus strains were found resistant to tigecycline, linezolid and daptomycin. VRE was found in Enterococcus faecium, accounting for 1.9% of all Enterococcus faecium strains.Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are the most common pathogens causing nosocomial infections. Nosocomial pathogens showed high susceptibilities against tigecycline. For ESBLs-producing Enterobacteriaceae strains, β-lactam+Inhibitor show high antibacterial activities. Vancomycin, teicoplanin and linezolid exhibit high potency to Staphylococcus aureus and Enterococcus.

To evaluate the long-term clinical therapeutic effect of polyaxial screw-rod system for posterior cervical arthrodesis on patients with upper cervical spinal cord tumors. From March 2007 to May 2013, 22 patients with upper cervical spinal cord tumors underwent tumor resection and posterior cervical arthrodesis in our institution. The medical records of these patients were reviewed respectively. There were 10 males and 12 females with ages ranging from 16 to 60 years old. Posterior cervical arthrodesis by polyaxial screw-rod was performed at the upper cervical spine (C1-3). All patients were followed-up clinically and radiographically. The average follow-up was 65.5 months. Twenty-two patients were enrolled and a total of 114 screws were placed in this study. Histopathology revealed neurinoma, meningioma, ganglioneuroma and ganglioglioma in 16, 3, 1 and 1 case (s), respectively. The mixed tumor with component of ganglioneuroma and neurinoma was observed in 1 case. All patients received tumor resection and posterior athrodesis by polyaxial screw-rod system. Cervical kyphosis was encountered in one patient and this patient suffered the recurrence of tumor. Solid fusion was achieved in all patients. The average postoperative Japanese Orthopaedic Association (JOA) score was 13.9 and the average recovery rate was 51.4%. Neurologic deterioration was found in 2 patients. No complications, such as spinal cord or vertebral artery injury, postoperative radiculopathy or instrumentation failure, were observed. The long-term clinical therapeutic effects of posterior cervical arthrodesis using polyaxial screw-rod system on upper cervical spinal cord tumors are satisfactory, with no severe complication.目的：评估钉棒系统用于颈椎后路关节融合术治疗上颈椎脊髓肿瘤患者的手术考量及长期疗效。方法： 回顾2007年3月至2013年5月因上颈髓肿瘤在中南大学湘雅二医院行肿瘤切除及颈椎后路关节融合术的22例患者的医 疗记录，其中包括男性10例，女性12例，年龄16~60岁。所有患者接受肿瘤切除，通过多轴钉棒系统对上颈椎(颈1~3) 进行后路关节融合术。所有患者接受平均5年的临床及影像学随访。结果：平均随访时间为65.5个月。22例患者共 置入114枚螺钉，组织病理学结果提示：神经鞘瘤16例，脊膜瘤3例，神经节细胞瘤1例，神经节神经胶质瘤1例，混 合肿瘤(成分包括神经节细胞瘤和神经鞘瘤)1例。所有病例均获成功融合，术后日本骨科协会( Japanese Orthopaedic Association，JOA)评分为13.9分，治愈率为51.4%。1例术后发生颈椎后凸和肿瘤复发，神经功能障碍加重2例。未发现 脊髓损伤、椎动脉损伤、术后神经根病、螺钉拔出或内固定失败等并发症。结论：通过多轴钉棒系统行颈椎后路关 节融合术用于治疗上颈椎脊髓肿瘤，能够获得满意的长期临床和影像学疗效，无严重并发症发生。.

To study the antibiotic resistance evolution, homology, phenotypes and genotypes of carbapenemase in Acinetobacter baumannii from clinical isolates.A total of 72 strains of Acinetobacter baumannii were isolated from Xiangya Hospital of Central South University from March to May 2012. Antimicrobial susceptibility test was carried out by automatic microorganism clinical analytical system VITEK-II. The homology of the 72 strains was analyzed by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Modified Hodge test was used to screen carbapenemases of the strains. Carbapenemase genes blaOXA-23, blaOXA-40 and blaOXA-58 were also amplified and sequenced.The 72 strains of Acinetobacter baumannii remained sensitive to cefoperazone/sulbactam (resistance rate 8.33%), followed by Amikacin. Otherwise, they were resistant to most of the antimicrobial agents (resistance rate more than 70%). The 72 strains were identified as 7 epidemic clones, A-G, by means of ERIC-PCR and the phylogenetic relationship among D, E, F and G was very close, suggesting a nosocomial infection possibility. Totally 56 strains produced carbapenemase; 61 strains were positive for carbapenemase gene blaOXA-23 and 1 strain positive for blaOXA-58. All strains were negative for carbapenemase gene blaOXA-40.Acinetobacter baumannii strains isolated clinically are resistant to most of the antimicrobial agents and nosocomial infection had been observed. Most of the strains produce carbapenemase, among which, blaOXA-23 gene is the main carbapenemase gene. blaOXA-58 gene exists in the Acinetobacter baumannii isolates from Hunan Province.

To survey antibiotic resistance of clinical isolates of Acinetobacter baumannii in Changsha and to investigate molecular epidemiological characteristics of carbapenem-resistant Acinetobacter baumannii.A total of 205 non-duplicated, clinical isolates of Acinetabacter baumannii from 10 general hospitals in Changsha were collected from March 2010 to December 2010. The K-B disk diffusion method was applied for the drug-susceptibility test; a modified, double-disk synergy test was used to detect metallo-β-lactamase (MBL), and a modified Hodge test was used for the screening of carbapenemase. PCR was used to amplify carbapenemase genes (including OXA-23, OXA-24, OXA-51, IMP-1, and VIM-2) and the positive products were sequenced. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) was used for DNA typing and test of homology.Of the 18 antibiotics tested, 14 had a high rate of resistance (50% of the isolates tested), with piperacillin the highest (80.5% of strains), and cefoperazone/sulbactam the lowest (2.5%). In total, 115 carbapenem-resistant Acinetobacter baumannii strains were confirmed, but their MBL phenotype and genes were all negative. Seventy-one positive strains were detected by the modified Hodge test, among which 64 strains were OXA-23-positive. All the 115 strains were positive for the amplification of the OXA-51 gene, and no strain was found which carried OXA-24 or OXA-58 gene. Seven genomic types were included in the 115 Acinetobacter baumannii. The major prevalence types were Type B ( 72 strains) and Type A (19 strains).Multiple drug resistance of clinically isolated Acinetobacter baumannii is a serious problem in Changsha. Production of OXA-23 and OXA-51 carbapenemases is an important mechanism of resistance to carbapenem antibiotics, and there is prevalence of the same clones in these carbapenem-resistant strains.

Carbapenem-resistant Acinetobacter baumannii has been known as an opportunistic pathogen that causes a variety of illness worldwide. In this study, we characterize the molecular epidemiology of 174 non-repetitive clinical isolates of A. baumannii collected from January to June 2009 from Xiangya Hospital, in Hunan Province, China, including an outbreak period of A. baumannii. These 174 isolates harbored A. baumannii intrinsic gene OXA-51. They were resistant to multiple antibiotics with resistance rates as 49.4% to imipenem, 48.3% to meropenem, 46.6% to ampicillin-sulbactam, 6.9% to cefoperazone-sulbactam, and 6.3% to minocycline. 74 out of 174 isolates were identified as carbapenemase-producing strains, among which bla(OXA-23) gene was found in 71 isolates. These 74 carbapenemase expression strains could be divided into four genotypes by enterobacterial repetitive intergenic consensus (ERIC)-PCR, with 19, 17, 33 and 5 clones in each group. We also found four imipenem resistant isolates carrying OXA-23 gene without showing carbapenemase phenotype. Our findings show that the bla(OXA-23) gene is the common carbapenemase gene among carbapenem-resistant Acinetobacter spp. isolates, suggesting that clonal spread of carbapenemase-producing isolates may be one important factor which results in the high carbapenem resistance rate in the local hospital in Hunan Province, China.

Objectives The aim of this study was to undertake a survey of the occurrence of CTX-M and SHV extended-spectrum beta-lactamase (ESBL) genotypes in Enterobacteriaceae from Hunan Province, China. Methods Clinical isolates (425) from three major hospitals in Changsha, Hunan Province, were collected between October 2004 and July 2005, and their antimicrobial susceptibilities of the genotype of bla(CTX-M) and bla(SHV) were determined. Random amplified polymorphic DNA was used to characterize the clonality of all of the isolates. Results The overall rate of ESBL-positive isolates was 33.4% (142/425). The dominant ESBLs were CTX-M types, and were found in 109/142 (76.8%) isolates comprising seven different genera/species, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris and Providencia stuartii. The most common bla(CTX-M) genotypes were bla(CTX-M-14) (47.7%), bla(CTX-M-3) (29.4%) and bla(CTX-M-15) (17.4%). A novel gene derived from bla(CTX-M-15), bla(CTX-M-82) (Ala-40-->Pro), was identified. Conclusions The dominant ESBL genotype in Hunan Province was bla(CTX-M). The high prevalence (17.4%) of bla(CTX-M-15) has not previously been reported from China. Our results identify that an epidemic of bla(CTX-M) in Changsha, Hunan Province, has evolved with the appearance and spread of bla(CTX-M-15) against the dominant genotypes bla(CTX-M-14) and bla(CTX-M-3.) The worldwide dominance of bla(CTX-M-15) could be poised to spread to China, displacing the current prevailing genotypes.