High Prevalence of Carbapenem-Resistant Enterobacterales Colonization Among Intensive Care Unit Patients in a Tertiary Hospital, China.

Intestinal colonization with carbapenem-resistant Enterobacterales (CRE) has been shown as a significant risk factor for subsequent CRE infections, especially in intensive care units (ICUs). The aim of this study was to determine the prevalence of intestinal CRE colonization among ICU patients in a Chinese tertiary hospital. Fecal sample screenings for CRE were performed on ICU patients weekly. Antibiotic-susceptibility profile of CRE strains was determined using the Vitek-2 analysis system and broth microdilution method. The carbapenemases of all isolates were determined by phenotypes and genotypes. Clonal relatedness was analyzed by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was used to identify the multilocus sequence type (ST), plasmid replicons, and insertion sequences (ISs) of isolates. The overall colonization rate of CRE was 40.4% (82/203). A total of 84 CRE strains were detected, mostly with Klebsiella pneumoniae (92.9%). Antibiotic susceptibility testing profile revealed that 84 CRE strains were resistant to most antibiotics except for tigecycline and colistin. The carbapenemase-encoding genes including blaKPC-2, blaNDM-1, and blaIMP-4 were detected, and blaKPC-2 was the predominant genotype (90.8%). A total of 9 STs were identified among 84 CRE strains, and ST11 was the most common type (83.3%). A variety of mobile genetic elements, including plasmids and ISs, were detected via online tool prediction. PFGE analysis of the 78 K. pneumoniae strains showed 8 different pulsotypes, and pulsotype A was highly prevalent. This study found that the prevalence of CRE colonization was alarmingly high in the ICU, and that effective infection control measures are urgently needed to prevent the dissemination of CRE.

Genetic and Functional Analysis of the pks Gene in Clinical Klebsiella pneumoniae Isolates.

The pks gene cluster encodes colibactin, which can cause DNA damage and enhance the virulence in Escherichia coli. However, the role of the pks gene in Klebsiella pneumoniae has not been fully discussed. The aim of this study was to analyze the relationship between the pks gene cluster and virulence factors, as well as to assess antibiotic resistance and biofilm formation capacity in clinical isolates of Klebsiella pneumoniae. Thirty-eight of 95 clinical K. pneumoniae strains were pks positive. pks-positive strains usually infected emergency department patients, and pks-negative strains often infected hospitalized patients. The positive rates of K1 capsular serotype and hypervirulence genes (peg-344, rmpA, rmpA2, iucA, and iroB) were significantly higher in the pks-positive isolates than the pks-negative isolates (P < 0.05). The biofilm formation ability of pks-positive isolates was stronger than that of pks-negative isolates. Antibacterial drug susceptibility test showed the resistance of pks-positive isolates was weaker than that of pks-negative isolates. In conclusion, patients with pks-positive K. pneumoniae infection might have worse treatment outcomes and prognosis. pks-positive K. pneumoniae might have stronger virulence and pathogenicity. Clinical infection with pks-positive K. pneumoniae needs further attention. IMPORTANCE The infection rate with pks-positive K. pneumoniae has been increasing in recent years. Two previous surveys in Taiwan reported 25.6% pks gene islands and 16.7% pks-positive K. pneumoniae strains in bloodstream infections, and Chinese scholars also did a survey of K. pneumoniae bloodstream infections in Changsha, China, and found 26.8% pks-positive K. pneumoniae. In addition, it was found that the pks gene cluster might encode colibactin, which could be related to the virulence of K. pneumoniae. Studies confirmed that the prevalence of colibactin-producing K. pneumoniae was increasing. It is necessary to consider the clear relationship between the pks gene cluster and high pathogenicity in K. pneumoniae.

Identification of hypervirulent Klebsiella pneumoniae carrying terW gene by MacConkey-potassium tellurite medium in the general population.

Objectives: To establish a MacConkey-potassium tellurium medium-based method for selectively culturing terW gene-positive Klebsiella pneumoniae (KP), to evaluate its performance and apply it to identifying particular clonal hypervirulent KP (hvKP) strains in epidemiological surveillance. Methods: The virulence genes, rmpA, iutA, and terW, were detected by PCR. The minimum inhibitory concentration of potassium tellurite of hvKP (rmpA +/ iutA +) and classical KP (rmpA - and iutA -) was determined using the agar dilution method. The MacConkey medium containing 4 µg/ml potassium tellurite was prepared and the performance in detecting terW + KP was evaluated, including an agreement with PCR and positive/negative predictive value. Fecal samples from healthy volunteers in Fujian were collected and cultured in the medium, then positive strains were identified using MALDI-TOF MS, antimicrobial susceptibility was tested by Kirby-Bauer assays, and virulence genes and capsular serotype genes were tested by PCR. Results: In KP isolated from clinical specimens (N = 198), the positive rate of terW was 37.9%, and the detection rate of terW in hvKP was significantly higher than that in classical KP (70.6% vs 13.3%). The potassium tellurite resistance levels of terW + (N = 75) and terW - (N = 55) KP were 8-128 µg/ml and <1-8 µg/ml, respectively, with significant differences. KP was selectively cultured on a MacConkey medium with 4 µg/ml potassium tellurite, and its agreement with PCR was good (Kappa=0.936), and the positive and negative percent agreement and positive and negative predictive values were 100% (75/75), 92.7% (51/55), 94.9% (75/79), and 100% (51/51), respectively. The prevalence of tellurite-resistant KP was 16.7% (86/516) in fecal samples from healthy volunteers, among which the positive rate of terW was 100% (86/86). The antimicrobial resistance characteristics of terW + KP showed no difference between healthy volunteers and inpatients. The most common capsular serotypes associated with high virulence were K1, K2, and K57. Conclusions: The MacConkey medium containing 4 µg/ml potassium tellurite could easily select and culture terW + KP in fecal samples with high sensitivity and specificity, which is a practical method for the epidemic surveillance of hvKP in the general population.

Gut microbiota dysbiosis in patients with hepatitis B virus-induced chronic liver disease covering chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.

The information regarding the effect of hepatitis B virus (HBV) infection on gut microbiota and the relationship between gut microbiota dysbiosis and hepatitis B virus-induced chronic liver disease (HBVCLD) is limited. In this study, we aimed at characterizing the gut microbiota composition in the three different stages of hepatitis B virus-induced chronic liver disease patients and healthy individuals. Faecal samples and clinical data were collected from HBVCLD patients and healthy individuals. The 16S rDNA gene amplification products were sequenced. Bioinformatic analysis including alpha diversity and PICRUSt was performed. A total of 19 phyla, 43 classes, 72 orders, 126 families and 225 genera were detected. The beta-diversity showed a separate clustering of healthy controls and HBVCLD patients covering chronic hepatitis (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC); and gut microbiota of healthy controls was more consistent, whereas those of CHB, LC and HCC varied substantially. The abundance of Firmicutes was lower, and Bacteroidetes was higher in patients with CHB, LC and HCC than in healthy controls. Predicted metagenomics of microbial communities showed an increase in glycan biosynthesis and metabolism-related genes and lipid metabolism-related genes in HBVCLD than in healthy individuals. Our study suggested that HBVCLD is associated with gut dysbiosis, with characteristics including, a gain in potential bacteria and a loss in potential beneficial bacteria or genes. Further study of CHB, LC and HCC based on microbiota may provide a novel insight into the pathogenesis of HBVCLD as well as a novel treatment strategy.

Toll-like receptors, long non-coding RNA NEAT1, and RIG-I expression are associated with HBeAg-positive chronic hepatitis B patients in the active phase.

BACKGROUND: Innate immunity plays a crucial role in host-virus interactions and greatly influences viral replication including HBV infection. However, few studies have investigated the possible antiviral immune roles played by TLRs, RIG-I, and long no-coding RNA NEAT1 in chronic HBV infection (CHB) patients in clinical samples and their relationships among immune responses. In this study, we sought to investigate the mRNA expression levels of TLR1-10, RIG-I, and NEAT1 expression in HBeAg-positive CHB treatment-naïve patients with the active phase. METHODS: The expression levels of TLR1-10, RIG-I, and NEAT1 of CHB patients with the active phase and healthy controls were measured by qPCR. Serum HBV DNA and routine liver biochemistry including ALT, etc were also measured to evaluate the impaired physiological function of the liver affected by CHB. RESULTS: The expression levels of TLR1 and TLR6 in CHB with active phase were remarkably lower than that in healthy controls. The levels of TLR3 in CHB patients with active phase were remarkably higher than that in healthy controls. The total NEAT1 expression was abnormally decreased in CHB patients as compared with healthy controls. The levels of RIG-I were significantly decreased in CHB patients in the active phase when compared to healthy controls. The expression of TLR6 and RIG-I was closely correlated with NEAT1 expression. TLR6 level was positively correlated with RIG-I level. CONCLUSION: Chronic HBV infection can alter the innate immune response by downregulating functional expression of TLR1, TLR6, NEAT1.