Antibiotic Resistances and Molecular Characteristics of Clostridioides difficile in ICUs in a Teaching Hospital From Central South China.

Clostridioides (C.) difficile is a major healthcare-associated pathogen inducing infectious diarrhea. Approximately 25-33% of patients with antibiotic-associated diarrhea (AAD) and 90% of patients with pseudomembranous enteritis are caused by C. difficile infection (CDI). Stool samples were collected from hospitalized adults with presumptive AAD in four nonneonatal intensive care units (ICUs). Diagnosis of CDI was based on both clinical symptoms and laboratory results. The stool specimens were transferred onto CDIF (C. difficile agar), and C. difficile was finally confirmed by the latex agglutination test. Toxin-producing genes tcdA (A), tcdB (B), and cdt (CDT) were detected by PCR, and all isolates were performed multilocus sequence typing analysis. The antibiotic susceptibility of C. difficile isolates was assessed by the agar dilution method. A total of 184 C. difficile were isolated from 857 specimens in our study, the isolation rate of C. difficile was 21.5% (184/857). The 184 C. difficile were isolated from 179 patients, among these 115 patients were toxin-positive, giving the incidence of CDI being 58.0/10,000 patient days in the four ICUs. Among these 115 toxin-positive C. difficile isolates, 100 (87.0%) isolates produced two toxins (A+B+CDT-), three (2.6%) isolates were A+B+ with binary toxin-producing (A+B+CDT+), and 12 (10.4%) isolates only produced one toxin (A-B+CDT-). A total of 27 sequencing types (STs) were obtained. The most prevalent was ST3 (34 isolates), followed by ST39 (27 isolates), ST54 (19 isolates), ST26 (16 isolates), ST35 (15 isolates), and ST2 (13 isolates). All the ST26 isolates were nontoxigenic. Meanwhile, five STs were newly discovered. Although multidrug resistance was present in ≥50% of these C. difficile isolates, all of them were susceptible to tigecycline, fidaxomicin, metronidazole, and vancomycin. In conclusion, C. difficile isolates producing two toxins (A+B+CDT-) were dominant in our hospital. The most prevalent was ST3, and all ST26 isolates were NTCD. Although multidrug resistance was present in ≥50% of the C. difficile isolates, metronidazole, tigecycline, fidaxomicin, and vancomycin were still effective treatments for CDI in our hospital.

Gut Microbiota Composition Associated With Clostridium difficile-Positive Diarrhea and C. difficile Type in ICU Patients.

The gut microbiota composition of intensive care unit (ICU) patients suffering from Clostridium difficile-positive diarrhea (CDpD) is poorly understood. This prospective study aims to use 16S rDNA (and metagenome) sequencing to compare the microbiota composition of 58 (and 5) ICU patients with CDpD (CDpD group), 33 (and 4) ICU patients with C. difficile-negative diarrhea (CDnD group), and 21 (and 5) healthy control subjects (control group), as well as CDpD patients in the A+B+ (N = 34; A/B: C. difficile TcdA/B), A-B+ (N = 7), and A-B- (N = 17) subgroups. For 16S rDNA data, OTU clustering (tool: UPARSE), taxonomic assignment (tool: RDP classifier), α-diversity, and ß-diversity analyses (tool: QIIME) were conducted. For metagenome data, metagenome assembly (tool: SOAPdenovo), gene calling (tools: MetaGeneMark, CD-HIT, and SoapAligner), unigene alignment (tool: DIAMOND), taxon difference analysis (tool: Metastats), and gene annotation (tool: DIAMOND) were performed. The microbial diversity of the CDpD group was lower than that of the CDnD and control groups. The abundances of 10 taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) were significantly higher in the CDpD group than in the CDnD group. The abundances of Saccharomycetes and Clostridia were significantly lower in CDpD in comparison with control. Some taxa were significantly different between the A+B+ and A-B- subgroups. CDpD might relate to a decrease in beneficial taxa (i.e., Saccharomycetes and Clostridia) and an increase in harmful taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) in gut microbiota of ICU patients. C. difficile toxin type might be slightly associated with gut microbiota composition.

Assessing Molecular Epidemiology of Carbapenem-resistant Klebsiella pneumoniae (CR-KP) with MLST and MALDI-TOF in Central China.

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.

Characterization of the virulence of a non-RT027, non-RT078 and binary toxin-positive Clostridium difficile strain associated with severe diarrhea.

The expression of the Clostridium difficile binary toxin CDT is generally observed in the RT027 (ST1) and RT078 (ST11) C. difficile isolates, which are associated with severe C. difficile infection (CDI). However, we recently reported that the non-RT027 and non-RT078 C. difficile strain LC693 (TcdA+TcdB+ CDT+, ST201) caused severe diarrhea in a patient in Xiangya Hospital in China. C.difficile LC693 is a member of Clade 3, and in this study, we identified LC693 as RT871 and compared its virulence and pathogenicity to those of C.difficile R20291 (TcdA+TcdB+CDT+, ST1/RT027), UK6 (TcdA+TcdB+CDT+, ST35/RT027), CD630 (TcdA+TcdB+CDT-, ST54, RT012), and 1379 (TcdA+TcdB+CDT-, ST54/RT012), with strain 1379 being an epidemic C.difficile isolate from the same hospital. LC693 displayed a higher sporulation rate than R20291, CD630 or strain 1379. LC693 was comparable to R20291 with respect to spore germination, motility, and biofilm formation, but showed a faster germination rate, higher motility and a higher biofilm formation capability compared to CD630 and strain 1379. The adherence of spores to human gut epithelial cells was similar for all strains.The total toxin release of LC693 was lower than that of R20291, but higher than that of CD630 and strain 1379. Finally, in a mouse model of CDI, LC693 was capable of causing moderate to severe disease. Our findings demonstrate the pathogenicity of non-RT027 and non-RT078 binary toxin-positive C. difficile strains. Furthermore, our data indicate that LC693 may be more virulent than strain 1379, an epidemic strain from the same hospital, and provide the first phenotypic characterization of a non-RT027 and non-RT078 binary toxin-positive ST201 isolate.

Incidence and Outbreak of Healthcare-Onset Healthcare-Associated Clostridioides difficile Infections Among Intensive Care Patients in a Large Teaching Hospital in China.

Background:Clostridioides difficile infection (CDI) is an important cause of morbidity and mortality among hospitalized patients. In China, however, hospital staff do not routinely test for CDI, leading to under-diagnosis and poor patient outcomes. Locally generated CDI data can help assess the magnitude of the problem and strengthen approaches for CDI prevention and control. Methods: We prospectively monitored hospital-onset hospital-associated (HOHA) CDI in four intensive care units (ICUs) from June 2013 to September 2014 in a large teaching hospital in China. We collected clinical information from all ICU patients with ≥ 3 episodes of diarrhea occurring within a 24-h period at least 48 h following admission (suspect case definition). Stool specimens were collected from all suspect cases of CDI and cultured for C. difficile. Polymerase chain reaction (PCR) was used to detect toxin genes from positive isolates; multi-locus sequence typing (MLST) was used for typing and identifying novel strains. We estimated the incidence rate as the number of HOHA CDI cases per 10,000 patient days; 95% confidence intervals were generated to assess rate differences between the four ICUs. Results: A total of 593 hospital-onset diarrhea patients met the suspect case definition during the study period. Of these, 47 patients (8%) were positive for C. difficile and toxin genes. The HOHA-CDI incidence rate was 14.1 cases per 10,000 patient days (95% CI: 10.5-18.6). Six patients with HOHA CDI died. ST54 (n = 14, 20%) was the most common type of HOHA-CDI strain circulating in the hospital during the study period and was linked to a temporal cluster (outbreak) involving two (NICU and GICU) of the four ICUs. Conclusion: HOHA-CDI occurs among ICU patients at this teaching hospital, supporting the importance of routine testing for CDI. Information on strain distribution can help detect CDI outbreaks. Detection of ST54 strain in a temporal cluster suggests possible gaps in infection control practices that should be investigated and addressed as needed.

Diagnosis of Clostridium difficile infection using an UPLC-MS based metabolomics method.

INTRODUCTION: The fecal metabolome of Clostridium difficile (CD) infection is far from being understood, particularly its non-volatile organic compounds. The drawbacks of current tests used to diagnose CD infection hinder their application. OBJECTIVE: The aims of this study were to find new characteristic fecal metabolites of CD infection and develop a metabolomics model for the diagnosis of CD infection. METHODS: Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was used to characterize the fecal metabolome of CD positive and negative diarrhea and healthy control stool samples. RESULTS: Diarrhea and healthy control samples showed distinct clusters in the principal components analysis score plot, and CD positive group and CD negative group demonstrated clearer separation in a partial least squares discriminate analysis model. The relative abundance of sphingosine, chenodeoxycholic acid, phenylalanine, lysophosphatidylcholine (C16:0), and propylene glycol stearate was higher, and the relative abundance of fatty amide, glycochenodeoxycholic acid, tyrosine, linoleyl carnitine, and sphingomyelin was lower in CD positive diarrhea groups, than in the CD negative group. A linear discriminant analysis model based on capsiamide, dihydrosphingosine, and glycochenodeoxycholic acid was further constructed to identify CD infection in diarrhea. The leave-one-out cross-validation accuracy and area under receiver operating characteristic curve for the training set/external validation set were 90.00/78.57%, and 0.900/0.7917 respectively. CONCLUSIONS: Compared with other hospital-onset diarrhea, CD diarrhea has distinct fecal metabolome characteristics. Our UPLC-MS metabolomics model might be useful tool for diagnosing CD diarrhea.

Genome characterization of a novel binary toxin-positive strain of Clostridium difficile and comparison with the epidemic 027 and 078 strains.

BACKGROUND: Clostridium difficile is an anaerobic Gram-positive spore-forming gut pathogen that causes antibiotic-associated diarrhea worldwide. A small number of C. difficile strains express the binary toxin (CDT), which is generally found in C. difficile 027 (ST1) and/or 078 (ST11) in clinic. However, we isolated a binary toxin-positive non-027, non-078 C. difficile LC693 that is associated with severe diarrhea in China. The genotype of this strain was determined as ST201. To understand the pathogenesis-basis of C. difficile ST201, the strain LC693 was chosen for whole genome sequencing, and its genome sequence was analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. RESULTS: The project finally generated an estimated genome size of approximately 4.07 Mbp for strain LC693. Genome size of the three ST201 strains ranged from 4.07 to 4.16 Mb, with an average GC content between 28.5 and 28.9%. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance. The ST201 strains contained a larger PaLoc with a Tn6218 element inserted than the 027/ST1 and 078/ST11 strains, and encoded a truncated TcdC. In addition, the ST201 strains contained intact binary toxin coding and regulation genes which are highly homologous to the 027/ST1 strain. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The presence of those genes also contributes to the pathogenesis of the ST201 strains. CONCLUSIONS: In this study, the genomic characterization of three binary toxin-positive C. difficile ST201 strains in clade 3 was discussed and compared to the genomes of the epidemic 027 and the 078 strains. Our analysis identified a number fitness and virulence associated genes/loci in the ST201 genomes that contribute to the pathogenesis of C. difficile ST201.

Assessing the risk and disease burden of Clostridium difficile infection among patients with hospital-acquired pneumonia at a University Hospital in Central China.

PURPOSE: Hospital-acquired pneumonia (HAP) remains one of the major hospital-acquired infections in China. Antibiotic treatment of HAP may lead to subsequent Clostridium difficile infection (CDI). Baseline data on the occurrence of CDI among HAP patients in China are currently unavailable. This study examines the risk and disease burden of CDI among HAP hospitalized patients (HAP-CDI). METHODS: We conducted a prospective study among ICU patients with HAP and hospital-onset diarrhea from January 2014 to December 2014 in a teaching hospital in China. All stool specimens were cultured for C. difficile which were typed by MLST. We used univariate and multivariable regression analyses to identify risk factors of HAP-CDI. FINDINGS: In total, 369 patients who met the inclusion criteria were enrolled. Thirty-two patients tested C. difficile positive. Among the isolated C. difficile strains, 90.63% (29/32) isolates were toxinogenic. Various MLST types were identified. The incidence of HAP-CDI was 11.67/10,000 patient days (95% CI, 7.97-16.55). Nineteen patients died from complications. The attributable mortality rate was 5.15% (19/369). The mortality rate of HAP-CDI group was 13.79% which was higher than HAP-non-CDI group. Univariate analyses demonstrated that old age, receiving antibiotics (OR = 8.70) and glucocorticoids (OR = 7.71) 1 month prior to hospitalization, respiratory failure (OR = 3.28) and receiving antimicrobials during hospitalization (OR = 1.15) were the risk factors associated with CDI. Multivariate conditional logistic regression analysis demonstrated the similar results. CONCLUSION: CDI was common among patients discharged from hospital for HAP at a university hospital. Prevention of the spreading of C. difficile among hospitalized patients is urgently needed.

Risk factors and medical costs for healthcare-associated carbapenem-resistant Escherichia coli infection among hospitalized patients in a Chinese teaching hospital.

BACKGROUND: The emergence and spread of Carbapenem-resistant Escherichia coli (CREC) is becoming a serious problem in Chinese hospitals, however, the data on this is scarce. Therefore, we investigate the risk factors for healthcare-associated CREC infection and study the incidence, antibiotic resistance and medical costs of CREC infections in our hospital. METHODS: We conducted a retrospective, matched case-control-control, parallel study in a tertiary teaching hospital. Patients admitted between January 2012 and December 2015 were included in this study. For patients with healthcare-associated CREC infection, two matched subject groups were created; one group with healthcare-associated CSEC infection and the other group without infection. RESULTS: Multivariate conditional logistic regression analysis demonstrated that prior hospital stay (<6 months) (OR:3.96; 95%CI:1.26-12.42), tracheostomy (OR:2.24; 95%CI: 1.14-4.38), central venous catheter insertion (OR: 8.15; 95%CI: 2.31-28.72), carbapenem exposure (OR: 12.02; 95%CI: 1.52-95.4), urinary system disease (OR: 16.69; 95%CI: 3.01-89.76), low hemoglobin (OR: 2.83; 95%CI: 1.46-5.50), and high blood glucose are associated (OR: 7.01; 95%CI: 1.89-26.02) with CREC infection. Total costs (p = 0.00), medical examination costs (p = 0.00), medical test costs (p = 0.00), total drug costs (p = 0.00) and ant-infective drug costs (p = 0.00) for the CREC group were significantly higher than those for the no infection group. Medical examination costs (p = 0.03), total drug costs (p = 0.03), and anti-infective drug costs (p = 0.01) for the CREC group were significantly higher than for the CSEC group. Mortality in CREC group was significantly higher than the CSEC group (p = 0.01) and no infection group (p = 0.01). CONCLUSION: Many factors were discovered for acquisition of healthcare-associated CREC infection. CREC isolates were resistant to most antibiotics, and had some association with high financial burden and increased mortality.

In vivo pharmacokinetics comparisons of icariin, emodin and psoralen from gan-kang granules and extracts of herba Epimedii, Nepal dock root, Ficus hirta yahl.

ETHNOPHARMACOLOGICAL RELEVANCE: Gan-kang granules (GKG) contains the extracts from eight Chinese herbs and is a traditional Chinese composite prescription for treatment of hepatitis B. Icariin, emodin and psoralen are main effective ingredients of the medicine. AIM OF THE STUDY: In this research, pharmacokinetic comparisons of icariin, emodin and psoralen from the extracts of herba Epimedii, Nepal dock root and Ficus hirta yahl, and GKG were conducted. MATERIALS AND METHODS: At different time points (0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 h) after administration, the concentrations of icariin, emodin and psoralen in rat plasma were determined by HPLC-UV, and main pharmacokinetic parameters were estimated. RESULTS: The pharmacokinetic parameters of icariin, emodin and psoralen in GKG were elevated comparing with those of herb extracts. CONCLUSIONS: Three HPLC-UV methods were developed successfully for the analysis of icariin, emodin and psoralen in SD rat plasma. Some ingredients in GKG may increase the dissolution and absorption, and improve bioavailability of icariin, emodin and psoralen in rats.

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.

A critical role of CCR7 in invasiveness and metastasis of SW620 colon cancer cell in vitro and in vivo.

Tumor cell migration and metastasis are critically regulated by chemokines and their receptors. CC Chemokine Receptor 7 (CCR7) plays a critical role in mediating chemotactic and invasive responses in cancers. However, whether or not CCR7 is a desired target of cancer therapy needs further investigation in terms of its biodegradation and availability in vivo. In this study, we employed RNA interference technology to detect the in vitro effects of anti-CCR7 siRNAs on proliferation and invasiveness of SW620 cells. We also evaluated the ability of these siRNAs to inhibit the lymphogenesis and the lymph node metastasis in xenografted SW620 tumor mouse. The chemotaxis and invasion assay in the animal model showed that blocking CCR7 expression at the mRNA level by a siRNA impaired invasion of colon cancer cells and inhibited lymph node metastasis of colon cancer and lymphogenesis. Therefore, CCR7 might be a desired target for cancer therapy and novel drug development.

Subchronic toxicity studies of Radix Astragali extract in rats and dogs.

Radix Astragali extract (RAE) is obtained from Astragalus membranaceus. It consists of Astragalus polysaccharide and Astragalus membranaceus saponins. In the study, we observed the subchronic toxicity of RAE in Sprague-Dawley rats and beagle dogs to evaluate the safety dosage range in clinical application. These subjects were daily administered of RAE by intra-peritoneum or vein for three consecutive months. General index were observed such as food-intake, behavior, body weight, hematological parameters, etc. Body weight, the weight of principal organ and hematology index are normal in experimental groups and control groups. The hematological biochemistry examination and histopathology examination of experimental groups are similar to control groups. In conclusion, our studies clearly demonstrated that RAE was safe without any distinct toxicity and side effects, the safety dosage range is 5.7-39.9g/kg for rats and 2.85-19.95g/kg for beagle dogs, which is equal to 70 or 35 times of that of human (0.57g/kg, say, average BW 70kg), respectively.