Yawei Zhang

High prevalence of hypervirulent Klebsiella pneumoniae infection in China: geographic distribution, clinical characteristics and antimicrobial resistance

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKP) is traditionally defined by hypermucoviscosity, but data based on genetic background are limited. Antimicrobial-resistant hvKP has been increasingly reported but has not yet been systematically studied. K. pneumoniae isolates from bloodstream infections, hospital-acquired pneumonia, and intra-abdominal infections were collected from 10 cities in China during February to July 2013. Clinical data were collected from medical records. All K. pneumoniae isolates were investigated by antimicrobial susceptibility testing, string test, extended-spectrum β-lactamase (ESBL) gene detection, capsular serotypes, virulence gene profiles, and multilocus sequence typing. hvKP was defined by aerobactin detection. Of 230 K. pneumoniae isolates, 37.8% were hvKP. The prevalence of hvKP varied among different cities, with the highest rate in Wuhan (73.9%) and the lowest in Zhejiang (8.3%). Hypermucoviscosity and the presence of K1, K2, K20, and rmpA genes were strongly associated with hvKP (P < 0.001). A significantly higher incidence of liver abscess (P = 0.026), sepsis (P = 0.038), and invasive infections (P = 0.043) was caused by hvKP. Cancer (odds ratio [OR], 2.285) and diabetes mellitus (OR, 2.256) appeared to be independent variables associated with hvKP infections by multivariate analysis. Importantly, 12.6% of hvKP isolates produced ESBLs, and most of them carried blaCTX-M genes. Patients with neutropenia (37.5% versus 5.6%; P = 0.020), history of systemic steroid therapy (37.5% versus 5.6%; P = 0.020), and combination therapy (62.5% versus 16.7%; P = 0.009) were more likely to be infected with ESBL-producing hvKP. The prevalence of hvKP is high in China and has a varied geographic distribution. ESBL-producing hvKP is emerging, suggesting an urgent need to enhance clinical awareness, especially for immunocompromised patients receiving combination therapy.

Genetic characterisation of clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline: Role of the global regulator RamA and its local repressor RamR.

Laboratory-derived Klebsiella pneumoniae mutants demonstrated that the ramA locus mediated low-level tigecycline resistance. The aim of this study was to elucidate the underlying mechanisms of tigecycline resistance in clinical K. pneumoniae isolates. In total, 106 isolates with tigecycline MICs ranging from 0.125 mg/L to 16 mg/L were collected to determine the correlations between expression of the global regulon ramA, marA, soxS, the acrB pump gene and tigecycline MICs. PCR was used to determine whether mutations in ramR, acrR or the rpsJ gene encoding 30S ribosomal protein S10 were responsible for tigecycline resistance. ramA or ramR inactivation and corresponding trans-complemented strains were used to characterise the contribution of RamA and RamR to tigecycline resistance. Tigecycline MICs were correlated with transcriptional levels of ramA and acrB, but were negatively correlated with marA and soxS. Disrupting ramA strikingly reduced the tigecycline MIC by 16-fold, accompanied by a 0.5-fold downregulation of acrB expression and 3.14- and 3.80-fold upregulation of marA and soxS, respectively. Complementation with plasmid-borne ramA restored the original parental phenotype of decreased tigecycline susceptibility. Of 34 tigecycline-non-susceptible isolates, 21 harbouring diverse mutations in RamR led to ramA overexpression. Disrupting the mutated ramR gene and complementing the mutated ramR gene with a wild-type gene downregulated expression of ramA but maintained the same tigecycline-resistant phenotype as the parental strain; the complemented strain exhibited 4.21- and 27.51-fold increased expression of acrB and marA, respectively. In conclusion, for the majority of tigecycline-resistant K. pneumoniae, ramA, depressed by ramR, was the major factor accounting for tigecycline resistance.

Efficacy and safety of daptomycin for the treatment of infectious disease: a meta-analysis based on randomized controlled trials

OBJECTIVES A systematic review and meta-analysis based on randomized controlled trials (RCTs) of the efficacy and safety of daptomycin versus comparators. METHODS Electronic databases (PubMed, Embase, Cochrane Central Register of Controlled Trials and clinical registered trials) were searched to identify RCTs that assessed the efficacy and safety of daptomycin versus therapy with comparators. Two reviewers independently applied selection criteria, performed a quality assessment and extracted the data. The I(2) statistic was calculated for heterogeneity, and a random-effects or fixed-effects model was used for estimates of risk ratio (RR). The primary outcome assessed was clinical treatment success among the intention-to-treat (ITT) population. RESULTS Thirteen trials fulfilled the inclusion criteria. Daptomycin was as efficacious as comparator regimens among the ITT population (RR = 0.98, 95% CI 0.93-1.03) but had a lower efficacy among the clinically evaluable (CE) population (RR = 0.96, 95% CI 0.93-1.00). Subgroup analyses according to the quality of the trial, the type of antibiotic and the type of infection did not alter the outcomes. No significant difference was identified for all-cause mortality between the daptomycin and comparator groups (RR = 1.17, 95% CI 0.76-1.79) but daptomycin therapy did reduce the duration of treatment. Daptomycin caused a significantly lower incidence of renal impairment, nausea and headache but caused a reversible increase in creatine phosphokinase (CPK). Subgroup analysis indicated that daptomycin was significantly associated with a higher incidence of CPK elevation and fewer renal impairments among the population with a mean age ≤60 years and a dose of daptomycin ≥6 mg/kg/24 h. CONCLUSIONS Daptomycin showed efficacy similar to the comparator regimens among the ITT population but lower efficacy among the CE population. Fewer adverse effects in total, but more CPK elevation effects, were observed in patients treated with daptomycin.

Evolution of Carbapenem-Resistant Acinetobacter baumannii Revealed through Whole-Genome Sequencing and Comparative Genomic Analysis

ABSTRACT Acinetobacter baumannii is a globally important nosocomial pathogen characterized by an evolving multidrug resistance. A total of 35 representative clinical A. baumannii strains isolated from 13 hospitals in nine cities in China from 1999 to 2011, including 32 carbapenem-resistant and 3 carbapenem-susceptible A. baumannii strains, were selected for whole-genome sequencing and comparative genomic analysis. Phylogenetic analysis revealed that the earliest strain, strain 1999BJAB11, and two strains isolated in Zhejiang Province in 2004 were the founder strains of carbapenem-resistant A. baumannii. Ten types of AbaR resistance islands were identified, and a previously unreported AbaR island, which comprised a two-component response regulator, resistance-related proteins, and RND efflux system proteins, was identified in two strains isolated in Zhejiang in 2004. Multiple transposons or insertion sequences (ISs) existed in each strain, and these gradually tended to diversify with evolution. Some of these IS elements or transposons were the first to be reported, and most of them were mainly found in strains from two provinces. Genome feature analysis illustrated diversified resistance genes, surface polysaccharides, and a restriction-modification system, even in strains that were phylogenetically and epidemiologically very closely related. IS-mediated deletions were identified in the type VI secretion system region, the csuE region, and core lipooligosaccharide (LOS) loci. Recombination occurred in the heme utilization region, and intrinsic resistance genes (blaADC and blaOXA-51-like variants) and three novel blaOXA-51-like variants (blaOXA-424, blaOXA-425, and blaOXA-426) were identified. Our results could improve the understanding of the evolutionary processes that contribute to the emergence of carbapenem-resistant A. baumannii strains and help elucidate the molecular evolutionary mechanism in A. baumannii.

Molecular epidemiology of colistin-resistant Enterobacteriaceae in inpatient and avian isolates from China: high prevalence of mcr-negative Klebsiella pneumoniae

Whether chromosomal and transmissible mechanisms contribute simultaneously to colistin resistance in Klebsiella pneumoniae and Escherichia coli remains unknown. This study aims to identify the underlying mechanisms of colistin resistance in inpatient and avian K. pneumoniae and E. coli in China. We retrospectively screened 2353 Enterobacteriaceae isolates from inpatients at multiple centers during 2011-2014, and 168 avian isolates from one slaughterhouse in 2013 for the presence of MCR-1/MCR-2. Mutations and transcriptional levels of the chromosomal RamA, PhoPQ, and PmrAB genes were determined by PCR and RT-qPCR. The transferability and genetic characteristics of the underlying colistin-resistance genes were detected by conjugation and whole-genome sequencing. The MIC90 for colistin in colistin-resistant K. pneumoniae (ColRKP, 128 mg/L, N = 17) was 16-fold higher than in colistin-resistant E. coli (ColREC, 8 mg/L, N = 33). The dominant sequence types of ColRKP were ST2018 and ST37, whereas ColREC displayed diversity. The chromosomal genes ramA, pmrB, and phoQ were not associated with colistin resistance in ColRKP. The transcriptional levels of PmrB in ColREC were 7.5-fold greater than in colistin-susceptible isolates. The carrying rates of MCR-1 in ColREC and ColRKP were 100% (33/33) and 23.5% (4/17), respectively. Plasmid IncI2 (~60 kb) carrying MCR-1 could be transferred to recipient E. coli EC600 with frequencies ranging from 8.74 × 10-6 to 1.31 × 10-4. No transferable genes were identified in mcr-1-negative ColRKP. MCR-1 combined with upregulated PmrB was associated with low-level colistin resistance in ColREC. However, two-thirds of the ColRKP isolates were mcr-negative and need to be studied further.

Decreased Fitness and Virulence in ST10 Escherichia coli Harboring blaNDM-5 and mcr-1 against a ST4981 Strain with blaNDM-5.

Although coexistence of blaNDM-5 and mcr-1 in Escherichia coli has been reported, little is known about the fitness and virulence of such strains. Three carbapenem-resistant Escherichia coli (GZ1, GZ2, and GZ3) successively isolated from one patient in 2015 were investigated for microbiological fitness and virulence. GZ1 and GZ2 were also resistant to colistin. To verify the association between plasmids and fitness, growth kinetics of the transconjugants were performed. We also analyzed genomic sequences of GZ2 and GZ3 using PacBio sequencing. GZ1 and GZ2 (ST10) co-harbored blaNDM-5 and mcr-1, while GZ3 (ST4981) carried only blaNDM-5. GZ3 demonstrated significantly more rapid growth (P < 0.001) and overgrew GZ2 with a competitive index of 1.0157 (4 h) and 2.5207 (24 h). Increased resistance to serum killing and mice mortality was also identified in GZ3. While GZ2 had four plasmids (IncI2, IncX3, IncHI2, IncFII), GZ3 possessed one plasmid (IncFII). The genetic contexts of blaNDM-5 in GZ2 and GZ3 were identical but inserted into different backbones, IncX3 (102,512 bp) and IncFII (91,451 bp), respectively. The growth was not statistically different between the transconjugants with mcr-1 or blaNDM-5 plasmid and recipient (P = 0.6238). Whole genome sequence analysis revealed that 28 virulence genes were specific to GZ3, potentially contributing to increased virulence of GZ3. Decreased fitness and virulence in a mcr-1 and blaNDM-5 co-harboring ST10 E. coli was found alongside a ST4981 strain with only blaNDM-5. Acquisition of mcr-1 or blaNDM-5 plasmid did not lead to considerable fitness costs, indicating the potential for dissemination of mcr-1 and blaNDM-5 in Enterobacteriaceae.

Transcriptional profiling of the two-component regulatory system VraSR in Staphylococcus aureus with low-level vancomycin resistance

The objective of this study was to comprehensively identify the target genes regulated by the two-component regulatory system VraSR in Staphylococcus aureus and to clarify the role of VraSR in low-level vancomycin resistance. Expression of vraS was determined by real-time quantitative reverse transcriptase PCR (qRT-PCR). A clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain B6D and a vancomycin-intermediate S. aureus (VISA) strain D7 that was induced from a meticillin-resistant S. aureus strain were selected to construct vraSR null mutants by allelic replacement. The vraSR-complemented strain B6D_c was also constructed by allelic replacement. Genes differentially expressed in the wild-type, vraSR null mutant and complemented strains were detected using RNA-Seq and were validated by qRT-PCR. Compared with vancomycin-susceptible S. aureus strains, expression of vraS was upregulated in all four isogenic hVISA strains. Vancomycin minimum inhibitory concentrations (MICs) in the vraSR null mutants B6D-ΔvraSR and D7-ΔvraSR were significantly lower than in the wild-type strains B6D and D7 and the complemented strain B6D_c. RNA-Seq and qRT-PCR data showed that expression of genes encoding FmtA protein, foldase protein PrsA, capsular polysaccharide biosynthesis glycosyltransferase, TcaA, a putative membrane protein, and six hypothetical proteins was down regulated in both vraSR-null mutants B6D-ΔvraSR and D7-ΔvraSR. Most of these differentially expressed proteins are involved in cell wall biosynthesis, which is associated with vancomycin resistance in S. aureus. In conclusion, VraSR plays an important role in S. aureus strains with low-level vancomycin resistance. PrsA, FmtA, glycosyltransferase and TcaA are regulated directly or indirectly by VraSR.

The role of RND efflux pump and global regulators in tigecycline resistance in clinical Acinetobacter baumannii isolates

AIM To analyze the expression and regulation of resistance-nodulation-division (RND) efflux systems in clinical tigecycline-nonsusceptible (TNS) Acinetobacter baumannii. MATERIALS & METHODS Comparisons of molecular and clinical characteristics were performed between 52 TNS and 53 tigecycline-susceptible isolates. Expression of RND efflux pumps and global regulators were analyzed by real-time RT-PCR. A complementation experiment was performed to evaluate the contribution of the adeRS mutations. RESULTS Mechanical ventilation and prior use of carbapenems were more common among patients with TNS strains. The relative expression of adeB and adeJ was increased significantly in TNS isolates. Complementarity to the adeR or adeS mutations decreased tigecycline susceptibility by ≤2-fold. Decreased expression of marR and soxR was detected in TNS isolates. CONCLUSION A correlation between tigecycline MIC and expression level of adeB and adeJ was identified. The influence of adeRS mutation on adeB expression was limited. Global regulators marR and soxR may be involved in tigecycline resistance.

Emergence of mcr-1 and carbapenemase genes in hospital sewage water in Beijing, China.

Objectives This study identified and characterized mcr-1-positive Enterobacteriaceae (MCRPE) and carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage water. Methods Influent and effluent sewage samples were collected from five tertiary hospitals in Beijing in December 2016. Samples were screened for MCRPE and CPE using antibiotic selection media. Results were confirmed by PCR amplification of β-lactamase and colistin resistance (mcr-1 and mcr-2) genes and by sequencing. Antimicrobial susceptibility testing, MLST, conjugation and plasmid typing and S1-nuclease-PFGE/Southern blotting were performed for all MCRPE and CPE isolates. Results Nine MCRPE and 12 CPE isolates were obtained. All mcr-1-positive isolates (n = 9) were Escherichia coli and belonged to eight different STs. The blaKPC-2-positive Enterobacteriaceae included Klebsiella pneumoniae (n = 4), Enterobacter cloacae (n = 4) and Citrobacter freundii (n = 1) isolates. Two C. freundii isolates and one E. cloacae isolate harboured the blaNDM-1 gene. MLST analysis revealed distinct genetic relatedness among all ST11 K. pneumoniae but not among any other carbapenemase-producing isolates. Conjugation and plasmid typing confirmed that three MCRPE isolates harboured mcr-1 on the self-transmissible IncX4 plasmid and the blaNDM-1 gene on the IncX3 plasmid. The sizes of the plasmids harbouring mcr-1, blaNDM-1 and blaKPC-2 were ∼33 to ∼240, ∼40 to ∼75 and ∼30 to ∼90 kb, respectively. Conclusions To the best of our knowledge, this is the first report of mcr-1-positive E. coli and blaNDM-1-carrying E. cloacae and C. freundii in hospital sewage water. These findings, especially the diversity of MCRPE and K. pneumoniae ST11 that harbour the blaKPC-2 gene, suggest that monitoring and management of hospital sewage water should be enhanced.

The prevalence of colistin resistance in escherichia coli and klebsiella pneumoniae isolated from food animals in China: coexistence of mcr-1 and bla NDM with low fitness cost

Increasing colistin resistance is a global concern because colistin is used as a last resort for the treatment of carbapenem-resistant Enterobacteriaceae infections. The plasmid-mediated colistin resistance gene, mcr-1 was found in distinct bacterial species isolated from humans, animals, and the environment. In this study, farms in four different agricultural provinces in China were investigated to determine the occurrence of the antimicrobial resistance and related genes. A total of 373 Escherichia coli and 54 Klebsiella pneumoniae were isolated from 510 non-duplicated samples. Of the E. coli and K. pneumoniae isolates, 72.7% and 66.7%, respectively, were susceptible to colistin. Isolates resistant to colistin comprised 46.6% of the samples isolated from Shandong, and 17.8% and 16.4% of the samples from Jilin and Henan, respectively. Twenty-six carbapenem-resistant E. coli isolates were resistant to colistin, in which both mcr-1 and blaNDM were present. Specifically, the co-existence was found in isolates from animals and sewage. Most of the resistance genes were located on plasmids and were 40-244 kilobases. Growth curves of transconjugants carrying mcr-1, blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-9 showed a low fitness cost compared with the recipient. In conclusion, mcr-1 was widespread in E. coli and K. pneumoniae isolated from farms in China. Co-existence of mcr-1 and blaNDM-9 was identified in different sequence types of E. coli with low fitness cost from various origins, indicating an urgent need to take measures for decreasing dissemination.