Emergence of colistin-heteroresistant and carbapenem-resistant hypervirulent Klebsiella pneumoniae.

OBJECTIVES: To investigate the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae, and characterize the underlying molecular mechanisms. METHODS: The population analysis profiles (PAPs) method was used to detect colistin heteroresistance. The time-killing assay was used to examine the effect of colistin on carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro. Galleria mellonella larvae infection model was used to test the potential virulence. qRT-PCR assay was conducted to compare the expression levels of efflux pump genes. Next and third-generation sequencing were conducted to analyse the genomic features. RESULTS: Two colistin-heteroresistant isolates were detected from a multi-center carbapenem-resistant Enterobacterales (CRE) surveillance study in China, which exhibited similar survival rates as the K2 hypervirulent reference strain ATCC 43816 in a G. mellonella larvae model. The two isolates belonged to ST11, harbouring the iucABCD, iutA, iroBCD, and rpmA2 hypervirulent genes and pLVPK-like virulence plasmids. Colistin showed a weak effect on the heteroresistant strains in vitro. The efflux pump genes acrA, acrB, tolC, oqxA, and oqxB were upregulated in this subpopulation compared to the parental strains. CONCLUSIONS: This study showed the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae. AcrAB-TolC and OqxAB efflux overexpression were involved in mediating colistin heteroresistance.

Genomic characterization of carbapenem-resistant Klebsiella oxytoca complex in China: a multi-center study.

Carbapenem-resistant (CR) Klebsiella oxytoca complex can be associated with high mortality, emerging as a new threat to the public health. K. oxytoca complex is phylogenetically close to K. pneumoniae, one of most common species associated with multidrug resistance in Enterobacterale. The latest research showed that K. oxytoca is a complex of six species. Currently, the epidemiological and genomic characteristics of CR K. oxytoca complex in China are still unclear. Here, we conducted a multi-center study on 25 CR K. oxytoca complex collected from five representative regions in China. These isolates were, respectively, recovered from respiratory tract (12 cases, 48.0%), abdominal cavity (5 cases, 20.0%), blood (4 cases, 16.0%), urine tract (3 cases, 12.0%) and skin or soft tissue (1 cases, 4.0%). Among them, 32.0% (8/25) of patients infected with K. oxytoca complex had a poor prognosis. In this study, three K. oxytoca complex species were detected, namely K. michiganensis, K. oxytoca and K. pasteurii, among which K. michiganensis was the most common. Three carbapenemase genes were identified, including blaNDM-1 (10, 38.5%), blaKPC-2 (9, 34.6%) and blaIMP (6 blaIMP-4 and 1 blaIMP-8; 7, 26.9%). Subsequent multilocus sequence typing identified various sequence types (STs), among which ST43, ST92 and ST145 were relatively common. Different from the clonal dissemination of high-risk carbapenem-resistant K. pneumoniae strains, our research revealed a polyclonal dissemination characteristic of CR K. oxytoca complex in China. S1-nuclease PFGE and Southern blot experiment showed that carbapenemase genes were encoded in plasmids of different sizes. Two blaNDM-harboring plasmids were subsequently sequenced, and were characterized to be IncX3 and IncC incompatibility groups, respectively. This is the first multi-center study of CR K. oxytoca complex in China, which improved our understanding of the prevalence and antimicrobial resistance characteristics of CR K. oxytoca complex in China.

Multicenter Study of Colistin Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae Strains in China.

Colistin has been considered a last-line option for the treatment of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Heterogeneous resistance to colistin leads to unexplained clinical colistin treatment failure for CRKP. Our study aimed to investigate the extent of colistin heteroresistance among CRKP strains in China. A total of 455 colistin-susceptible strains, collected from six tertiary care hospitals in China, were characterized. The overall rate of colistin heteroresistance was 6.2%, as determined by the population analysis profiles (PAPs). Genomic analysis revealed that 60.7% of the colistin-heteroresistant isolates belonged to the epidemic sequence type 11 (ST11) clone. Single-nucleotide polymorphisms (SNPs) suggested that 6 ST5216 strains shared the same origin. Each of the subpopulations had a ≥8-fold decrease in colistin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which indicated that heteroresistance could be suppressed by an efflux pump inhibitor. In addition, our results suggested that the PhoPQ pathway plays an important role in the mechanisms of heteroresistance. IMPORTANCE The problem of CRKP has raised alarms concerning global health. Our study enriches the epidemiological study of colistin heteroresistance among CRKP strains in China, where the prevalence of this phenomenon was previously unknown. Importantly, colistin-heteroresistant strains may cause the failure of clinical treatment with colistin, even if the clinical laboratory reports that the strains are sensitive. The commonly used broth microdilution method is unable to detect this special phenomenon. Additionally, our results indicate that efflux pumps play a major role in colistin heteroresistance, and inhibitors can effectively reverse it. Our study is the first to provide a detailed analysis of the prevalence of colistin heteroresistance in China, as well as an analysis of the genetic mechanisms of this phenomenon.

Acinetobacter baumannii adaptation to the host pH microenvironment is mediated by allelic variation in a single residue of BauA protein.

Acinetobacter baumannii has been listed as one of the most critical pathogens in nosocomial infections; however, the key genes and mechanisms to adapt to the host microenvironment lack in-depth understanding. In this study, a total of 76 isolates (from 8 to 12 isolates per patient, spanning 128 to 188 days) were longitudinally collected from eight patients to investigate the within-host evolution of A. baumannii. A total of 70 within-host mutations were identified, 80% of which were nonsynonymous, indicating the important role of positive selection. Several evolutionary strategies of A. baumannii to increase its potential to adapt to the host microenvironment were identified, including hypermutation and recombination. Six genes were mutated in isolates from two or more patients, including two TonB-dependent receptor genes (bauA and BJAB07104_RS00665). In particular, the siderophore receptor gene bauA was mutated in multiple isolates from four patients with three MLST types, and all mutations were at amino acid 391 in ligand-binding sites. With 391T or 391A, BauA was more strongly bound to siderophores, which promoted the iron-absorption activity of A. baumannii at acidic or neutral pH, respectively. Through the A/T mutation at site 391 of BauA, A. baumannii displayed two reversible phases to adapt to distinct pH microenvironments. In conclusion, we demonstrated the comprehensive within-host evolutionary dynamics of A. baumannii, and discovered a key mutation of BauA site 391 as a genetic switch to adapt to different pH values, which may represent a model in the pathogen evolutionary adaption of the host microenvironment.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter strains in China: a multicenter genomic study.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter (CR-Ent) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from Enterobacter-caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR-Ent strains collected from multiple tertiary health centres. The precise species of Enterobacter strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). Molecular features of high-risk CR-Ent sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR-Ent species in China was E. xiangfangensis (66/92, 71.93%), and the proportion of carbapenemase-producing Enterobacter (CP-Ent) in CR-Ent was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 E. xiangfangensis were the major lineages of CP-Ent strains, and ST171 E. xiangfangensis was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 E. xiangfangensis. In addition, the blaNDM-harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR-Ent strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR-Ent in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 E. xiangfangensis, and the blaNDM-harbouring IncX3-type plasmid were detected and emphasized.

Genomic characterization of a new phage BUCT541 against Klebsiella pneumoniae K1-ST23 and efficacy assessment in mouse and Galleria mellonella larvae.

Over the past decades, the spread of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) is becoming a new threat and new effective therapies against this pathogen are needed. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infections compared with antibacterial drug usage. Here, we reported a new phage BUCT541 which can lyse MDR-KP ST23. The genome of BUCT541 is a double-stranded linear 46,100-bp long DNA molecule with 48% GC content through the Next generation sequencing (NGS) data. A total of 81 open reading frames and no virulence or antimicrobial resistance genes are annotated in the BUCT541 genome. BUCT541 was able to lyse 7 of the 30 tested MDR-KP according to the host range analysis. And the seven sensitive strains belonged to the K. pneumoniae K1-ST23. BUCT541 exhibited high thermal stability (4-70°C) and broad pH tolerance (pH 3-11) in the stability test. The in vivo results showed that BUCT541 (4 × 105 plaque-forming units (PFU)/each) significantly increased the survival rate of K. pneumoniae infected Galleria mellonella from 5.3% to 83.3% within 48 h. Moreover, in the mouse lung infection model, high doses of BUCT541 (2 × 107 PFU/each) cured 100% of BALB/c mice that were infected with K. pneumoniae. After 30 h of treatment with phage BUCT541 of the multiplicity of infection (MOI) = 10, the K. pneumoniae in the lungs of mice was lower than 104 CFU/mL, compared to the control group 109 CFU/mL. Together, these findings indicate that phage BUCT541 holds great promise as an alternative therapy with excellent stability and a wide lysis range for the treatment of MDR-KP ST23 infection.

Capsule type defines the capability of Klebsiella pneumoniae in evading Kupffer cell capture in the liver.

Polysaccharide capsule is the main virulence factor of K. pneumoniae, a major pathogen of bloodstream infections in humans. While more than 80 capsular serotypes have been identified in K. pneumoniae, only several serotypes are frequently identified in invasive infections. It is documented that the capsule enhances bacterial resistance to phagocytosis, antimicrobial peptides and complement deposition under in vitro conditions. However, the precise role of the capsule in the process of K. pneumoniae bloodstream infections remains to be elucidated. Here we show that the capsule promotes K. pneumoniae survival in the bloodstream by protecting bacteria from being captured by liver resident macrophage Kupffer cells (KCs). Our real-time in vivo imaging revealed that blood-borne acapsular K. pneumoniae mutant is rapidly captured and killed by KCs in the liver sinusoids of mice, whereas, to various extents, encapsulated strains bypass the anti-bacterial machinery in a serotype-dependent manner. Using capsule switched strains, we show that certain high-virulence (HV) capsular serotypes completely block KC's capture, whereas the low-virulence (LV) counterparts confer partial protection against KC's capture. Moreover, KC's capture of the LV K. pneumoniae could be in vivo neutralized by free capsular polysaccharides of homologous but not heterologous serotypes, indicating that KCs specifically recognize the LV capsules. Finally, immunization with inactivated K. pneumoniae enables KCs to capture the HV K. pneumoniae. Together, our findings have uncovered that KCs are the major target cells of K. pneumoniae capsule to promote bacterial survival and virulence, which can be reversed by vaccination.

Clinical and Molecular Analysis of ST11-K47 Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae: A Strain Causing Liver Abscess.

Klebsiella pneumoniae has been the predominant pathogen of liver abscess, but ST11-K47 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has rarely been studied as the causative organism. We identified an ST11-K47 CR-hvKP (HvKp-su1) from the drainage fluid of a liver abscess in a Chinese man who was diagnosed with liver abscess combined with diabetes, pneumonia, pleural infection, abdominal abscess, and splenic abscess. HvKp-su1 was non-hypermucoviscous and lacked the magA and rmpA genes and pLVPK plasmid but exhibited high virulence, with a high mortality rate (90%) to wax moth larvae (G. mellonella), similar to the hypervirulent Klebsiella pneumoniae ATCC43816 (91.67%). Whole-genome sequencing and bioinformatics analysis indicated that HvKp-su1 possesses a plasmid similar to a type of pLVPK-like plasmid (JX-CR-hvKP-2-P2), which is an uncommon plasmid in CR-hvKP. HvKp-su1 carried multiple resistance genes, including blaKPC-2. blaTEM-1, blaSHV-55, and blaCTX-M-65; hypervirulence genes such as aerobactin (iutA), salmochelin (iroEN), and yersiniabactin (ybtAEPQSTUX); and the type 3 fimbriae-encoding system (mrkACDF). Moreover, v_5377 and v_5429 (cofT, CFA/III (CS8)) located on plasmid 1 were simultaneously predicted to be virulence genes. After the long-term combination use of antibiotics, the patient successfully recovered. In summary, our study clarified the clinical and molecular characteristics of a rare ST11-K47 CR-hvKP (HvKp-su1), raising great concerns about the emergence of ST11-K47 CR-hvKP with multidrug resistance and hypervirulence, and providing insights into the control and treatment of liver abscess caused by ST11-K47 CR-hvKP.

Polymyxin resistance in carbapenem-resistant Enterobacteriaceae isolates from patients without polymyxin exposure: a multicentre study in China.

Polymyxins were recently approved for the clinical treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections in China. The aim of this study was to determine the prevalence and molecular mechanisms of polymyxin-resistant CRE prior to the clinical application of polymyxin and to evaluate the potential for emerging polymyxin resistance in China. A total of 504 unique CRE isolates were collected from six tertiary-care hospitals in China between October 2016 and September 2017. All isolates underwent antimicrobial susceptibility testing. Clinical, demographic, antimicrobial exposure and infection data were collected from patients' medical charts. PCR detection, Sanger sequencing and reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) were used to investigate the molecular mechanism of polymyxin resistance. A total 19 (3.8%) polymyxin-resistant isolates were identified, including Klebsiella pneumoniae, Escherichia coli, Klebsiella aerogenes and Enterobacter cloacae. Genetic analysis in K. pneumoniae strains identified insertion sequence (IS) elements (n = 3), a stop codon (n = 1) and gene deletion (n = 2) in mgrB and a pmrB missense mutation (T157P) (n = 1). Two E. coli isolates contained mcr-1 and an E. cloacae strain harboured a frameshift in mgrB. Further transcriptional analysis showed that pmrA, pmrB, pmrC and pmrK were significantly upregulated in polymyxin-resistant isolates. Despite the lack of polymyxin exposure, 3.8% of CRE were resistant to polymyxin in China. Both chromosomal and plasmid-encoded mechanisms were identified. Our study suggests that clinical practice should be alert to pre-existing polymyxin resistance among CRE isolates to avoid further dissemination of polymyxin resistance.

Reduced Ceftazidime-Avibactam Susceptibility in KPC-Producing Klebsiella pneumoniae From Patients Without Ceftazidime-Avibactam Use History - A Multicenter Study in China.

KPC-producing Klebsiella pneumoniae (KPC-KP) is the most widely spread carbapenem-resistant Enterobacteriaceae (CRE) in China. Avibactam is a novel non-ß-lactam ß-lactamase inhibitor which is highly active against KPC. Recently, ceftazidime-avibactam (CAZ-AVI) was approved for clinical treatment in China. Here we conducted a retrospective study to examine the antimicrobial susceptibility of CAZ-AVI prior to its usage in China, and evaluated the potential to develop resistance in KPC-KP. CAZ-AVI MICs were tested in 347 KPC-KP isolates collected from patients with no prior treatment with this combination from six medical centers in China. Almost all isolates (n = 346; 99.7%) were CAZ-AVI-susceptible, with only 12 (3.5%) which showed reduced susceptibility (MIC ≥ 4/4 µg/ml) or resistance. The 12 isolates belong to ST11 and half of them carry virulence genes. In comparison to susceptible isolates, these isolates demonstrated higher bla KPC-2 copy numbers and expressions, and demonstrated higher frequency of developing CAZ-AVI resistance.

Multicenter Evaluation of the Xpert Carba-R Assay for Detection and Identification of Carbapenemase Genes in Sputum Specimens.

Rapid diagnosis of infections caused by carbapenem-resistant Enterobacteriaceae (CRE) is crucial for proper treatment and infection control. The Xpert Carba-R assay is a qualitative multiplex real-time PCR method that qualitatively detects and differentiates five common carbapenemase genes (blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP) directly from rectal swabs or purified colonies within approximately 1 h. We performed a multicenter evaluation of the investigational use of the Carba-R assay for detection and differentiation of carbapenemase genes from sputum specimens in patients with a clinical diagnosis of pneumonia. The intra- and interassay coefficients of variation values for the Carba-R assay were 0.2% to 2.0% and 1.4% to 2.3%, respectively. A total of 301 sputum specimens were collected and tested. Compared to bacterial culture followed by PCR identification of resistance genes from colonies, the Carba-R assay reduced turnaround time from 56 to 84 h to less than 2 h. Carbapenemase genes were detected by the Carba-R assay in Klebsiella pneumoniae (n = 236), Escherichia coli (n = 22), Enterobacter cloacae (n = 23), Klebsiella oxytoca (n = 8), Serratia marcescens (n = 6), Citrobacter freundii (n = 4), and Klebsiella aerogenes (n = 2). The Carba-R assay detected 112 blaKPC (33.5%), 70 blaNDM (21.0%), 8 blaIMP (2.4%), and 2 blaVIM (0.6%) genes, with positive percent agreement, negative percent agreement, and concordance rates of 92.9%, 86.7%, and 88.3%, respectively, for the dominant blaKPC and 85.0%, 87.8%, and 87.4%, respectively, for the blaNDM genes. Neither method detected the blaOXA-48 carbapenemase gene. The convenient, rapid, and simple characteristics of the Xpert Carba-R assay make it a potential tool for CRE detection and identification directly in sputum specimens.

In vitro activity of aztreonam-avibactam against metallo-ß-lactamase-producing Enterobacteriaceae-A multicenter study in China.

OBJECTIVES: To study the molecular epidemiology of clinical metallo-ß-lactamase (MBL)-producing Enterobacteriaceae isolates in China and to evaluate the antimicrobial susceptibility of MBL-Enterobacteriaceae isolates to aztreonam-avibactam. METHODS: Bacterial speciation was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCR was used to screen for common carbapenemase genes. Antimicrobial susceptibility testing of common clinical antibiotics and aztreonam-avibactam was performed using the standard broth microdilution method. RESULTS: A total of 161 MBL-Enterobacteriaceae isolates were included, with Klebsiella pneumoniae (n = 73, 45.4%) and Escherichia coli (n = 53, 32.9%) being the most common species. Among the 161 isolates, blaNDM (n = 151), blaIMP (n = 13), and blaVIM (n = 2) were detected, including five strains (3.1%) co-harboring two MBLs. MBL-Enterobacteriaceae isolates frequently contained two (n = 55, 34.2%) or more (n = 89, 55.3%) additional serine ß-lactamase genes (blaKPC, blaCTX-M, blaTEM, or blaSHV). Antimicrobial susceptibility testing showed that 81.4% of isolates (n = 131) were resistant to aztreonam. The rates of resistance to cefazolin, ceftazidime, ceftriaxone, cefotaxime, ampicillin-sulbactam, amoxicillin-clavulanic acid, and piperacillin-tazobactam were all over 90%. The addition of avibactam (4 µg/ml) significantly reduced the minimum inhibitory concentrations (MICs) of the aztreonam-resistant isolates by more than 8-fold (range ≤0.125 to 4 µg/ml), with a MIC50/MIC90 of ≤0.125/1 µg/ml among the 131 isolates. Overall, 96.9% (n = 156) of the total isolates were inhibited at an aztreonam-avibactam concentration of ≤1 µg/ml. Univariate and multivariate logistic regression analysis found that in patients with MBL-Enterobacteriaceae infections, the presence of pre-existing lung disease (adjusted odds ratio 8.267, 95% confidence interval 1.925-28.297; p = 0.004) was associated with a hazard effect on worse disease outcomes. CONCLUSIONS: The combined use of aztreonam-avibactam is highly potent against MBL-Enterobacteriaceae and may serve as a new candidate for the treatment of infections caused by MBL-Enterobacteriaceae in China.

[Progress in the diagnosis of carbapenem-resistant Enterobacteriaceae].

Carbapenem-resistant Enterobacteriaceae (CRE) is rising rapidly all over the world, and challenges clinical diagnosis and treatment by various genotypes. This paper summarizes the characteristics and diagnostic methods of CRE. CRE can be divided into three categories and five families with various characteristics and resistant genes. The diagnosis method include the Kirby-Bauer screening test, double disc synergetic inhibition test with carbapenems collaboration (double disc EDTA and meropenem, phenylboronic acid and meropenem), modified Hodge test, chromogenic medium detection for selected test of CRE, and Carba NP colorimetric test, PCR and sequencing for CRE confirmation test. Each method has its own advantages and disadvantages, and can be applied according to the local main popular CRE genetypes and experimental conditions.

Clinical and bacteriological features and prognosis of ascitic fluid infection in Chinese patients with cirrhosis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) and bacterascites (BA) represent frequent and serious complications in cirrhosis patients with ascites. However, few detailed data are available regarding the clinical and bacteriological feature of SBP or BA patients in China. METHODS: We retrospectively analyzed bacteriological and clinical characteristics of patients with SBP and BA at Beijing 302 Hospital in China from January 2012 to December 2015. RESULTS: A total of 600 patients with SBP (n = 408) or BA (n = 192) were enrolled. Patients with BA appeared to have a less severe clinical manifestation and lower mortality rate than patients with SBP. Gram-negative bacteria formed the majority of pathogens in SBP (73.9%) and BA (55.8%) cases. Higher ascitic fluid polymorphonuclear leucocytes (PMN) count and hepatocellular carcinoma were independent risk factors for BA episode progressing to SBP. The concentration of blood urea nitrogen (BUN) was independent risk factor for 30-day mortality of BA patients. For patients with SBP, the independent risk factors for 30-day mortality were age, Model for End-Stage Liver Disease (MELD) score, septic shock and hepatocellular carcinoma. Patients with third-generation cephalosporin or carbapenems resistant infection had a significantly lower survival probability. There were significant differences in clinical characteristics and outcome among the major bacteria. Multivariate analysis showed that patients infected with Klebsiella spp. had higher hazard ratio of 30-day mortality. CONCLUSION: Our study reported the bacteriological and clinical characteristics of patients with SBP and BA. Higher ascitic fluid PMN count and hepatocellular carcinoma were found to be independent risk factors for BA episode progressed to SBP. Outcome of ascitic fluid infection in patients with cirrhosis was influenced by the type of bacteria and antimicrobial susceptibility.

Elevated MIC Values of Imidazole Drugs against Aspergillus fumigatus Isolates with TR34/L98H/S297T/F495I Mutation.

The use of azole fungicides in agriculture is believed to be one of the main reasons for the emergence of azole resistance in Aspergillus fumigatus Though widely used in agriculture, imidazole fungicides have not been linked to resistance in A. fumigatus This study showed that elevated MIC values of imidazole drugs were observed against A. fumigatus isolates with TR34/L98H/S297T/F495I mutation, but not among isolates with TR34/L98H mutation. Short-tandem-repeat (STR) typing analysis of 580 A. fumigatus isolates from 20 countries suggested that the majority of TR34/L98H/S297T/F495I strains from China were genetically different from the predominant major clade comprising most of the azole-resistant strains and the strains with the same mutation from the Netherlands and Denmark. Alignments of sterol 14α-demethylase sequences suggested that F495I in A. fumigatus was orthologous to F506I in Penicillium digitatum and F489L in Pyrenophora teres, which have been reported to be associated with imidazole resistance. In vitro antifungal susceptibility testing of different recombinants with cyp51A mutations further confirmed the association of the F495I mutation with imidazole resistance. In conclusion, this study suggested that environmental use of imidazole fungicides might confer selection pressure for the emergence of azole resistance in A. fumigatus.

Epidemiology and antifungal susceptibilities of yeast isolates causing invasive infections across urban Beijing, China.

AIM: To investigate the species distribution and antifungal susceptibility profiles of yeast isolates causing invasive infections across Beijing. MATERIALS & METHODS: A total of 1201 yeast isolates recovered from blood and other sterile body fluids were correctly identified by matrix-assisted laser desorption/ionization TOF MS supplemented by DNA sequencing. Antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute broth microdilution method. RESULTS: Candida (95.5%) remained the most common yeast species isolated; Candida albicans (38.8%) and Candida parapsilosis (22.6%) were the leading species of candidemia. Azole resistances were mainly observed in Candida glabrata and Candida tropicalis isolates. CONCLUSION: This study outlined the epidemiologic data of invasive yeast infections and highlighted the need for continuous monitoring of azole resistances among C. glabrata and C. tropicalis isolates in Beijing.

Molecular epidemiology of bla OXA-23 -producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii poses a significant threat to hospitalized patients, as few therapeutic options remain. Thus, we investigated the molecular epidemiology and mechanism of resistance of carbapenem-resistant A.baumannii isolates in Beijing, China. METHODS: Carbapenem-resistant A.baumannii isolates (n = 101) obtained between June 2009 and November 2014 were used. Multilocus sequence typing (MLST) and PCR assays for class C and D ß-lactamase were performed on all isolates. S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to identify the resistance gene location. RESULTS: All 101 A.baumannii isolates were highly resistant to frequently used antimicrobials, and were considered multidrug resistant. A total of 12 sequence types (STs) were identified, including 10 reported STs and 2 novel STs. Eighty-seven isolates were classified to clonal complex 92 (CC92), among which ST191 and ST195 were the most common STs. The bla OXA-23 gene was positive in most (n = 95) of the A.baumannii isolates. Using S1-nuclease digestion PFGE and Southern blot hybridization, 3 patterns of plasmids carrying bla OXA-23 were confirmed. ST191 and ST195 (both harboring bla OXA-23 ) caused outbreaks during the study period, and this is the first report of outbreaks caused by ST191 and ST195 in north China. CONCLUSION: bla OXA-23 -producing A.baumannii ST191 and ST 195 isolates can disseminate in a hospital and are potential nosocomial outbreak strains. Surveillance of imipenem-resistant A.baumannii and antimicrobial stewardship should be strengthened.

Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China.

Azole resistance in Aspergillus fumigatus has emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance in A. fumigatus from different parts of China. A total of 317 clinical and 144 environmental A. fumigatus isolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility, cyp51A gene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmental A. fumigatus isolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in the cyp51A gene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in the cyp51A gene that produced amino acid changes among azole-susceptible A. fumigatus isolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistant A. fumigatus might be attributed to the environmental resistance mechanisms in China.

Allelic Variation of the Capsule Promoter Diversifies Encapsulation and Virulence In Streptococcus pneumoniae.

The polysaccharide capsule is the major virulence factor of Streptococcus pneumoniae (pneumococcus), a major human pathogen. The sequences in the promoter and coding regions of the capsule gene locus undergo extensive variations through the natural transformation-mediated horizontal gene transfer. The sequence variations in the coding region have led to at least 97 capsular serotypes. However, it remains unclear whether the sequence polymorphisms in the promoter region have any biological significance. In this study, we determined the sequences of the cps promoter region from 225 invasive pneumococcal isolates, and identified modular composition and remarkable inter-strain sequence variations in this region. The strain-to strain variations in the cps promoter are characterized by diversity in sequence and size, mosaic combinations of nucleotide polymorphisms and sequence modules, selective preservation of the sequence combinations, and promiscuous assortments of the sequences between the promoter and coding regions. Isogenic pneumococci carrying allelic variants of the cps promoter displayed significant differences in the transcription of the capsule genes, capsule production, adhesion to host epithelial cells, anti-phagocytosis and virulence in mouse bacteremia model. This study has thus indicated that the sequence polymorphisms in the cps promoter represent a novel mechanism for fine-tuning the level of encapsulation and virulence among S. pneumoniae strains.

Detection of the mcr-1 Colistin Resistance Gene in Carbapenem-Resistant Enterobacteriaceae from Different Hospitals in China.

The spread of the plasmid-mediated colistin resistance gene, mcr-1, into carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates poses a significant threat to global health. Here we report the identification of three mcr-1-harboring carbapenem-resistant Escherichia coli strains, collected from three patients in two provinces in China. Our results show that mcr-1-harboring CRE strains have started to spread in different hospitals in China. In addition, this report presents the first description of chromosomal integration of mcr-1 into a carbapenem-resistant E. coli strain.