Ying-Chun Xu

Molecular Epidemiology of Clinical Isolates of Carbapenem-Resistant Acinetobacter spp. from Chinese Hospitals

ABSTRACT Carbapenem resistance in Acinetobacter spp. is an emerging problem in China. We investigated the molecular epidemiology and carbapenemase genes of 221 nonrepetitive imipenem-resistant clinical isolates of Acinetobacter spp. collected from 1999 to 2005 at 11 teaching hospitals in China. Genotyping by pulsed-field gel electrophoresis (PFGE) found 15 PFGE patterns. Of these, one (clone P) was identified at four hospitals in Beijing and another (clone A) at four geographically disparate cities. Most imipenem-resistant isolates exhibited high-level resistance to all β-lactams and were only susceptible to colistin. blaOXA-23-like genes were found in 97.7% of isolates. Sequencing performed on 60 representative isolates confirmed the presence of the blaOXA-23 carbapenemase gene. Analysis of the genetic context of blaOXA-23 showed the presence of ISAba1 upstream of blaOXA-23. All of the 187 A. baumannii isolates identified by amplified RNA gene restriction analysis carried a blaOXA-51-like oxacillinase gene, while this gene was absent from isolates of other species. Sequencing indicated the presence of blaOXA-66 for 18 representative isolates. Seven isolates of one clone (clone T) carried the plasmid-mediated blaOXA-58 carbapenemase gene, while one isolate of another clone (clone L) carried the blaOXA-72 carbapenemase gene. Only 1 isolate of clone Q carried the blaIMP-8 metallo-β-lactamase gene, located in a class 1 integron. Of 221 isolates, 77.8% carried blaPER-1-like genes. Eleven different structures of class 1 integrons were detected, and most integrons carried genes mediating resistance to aminoglycosides, rifampin, and chloramphenicol. These findings indicated clonal spread of imipenem-resistant Acinetobacter spp. and wide dissemination of the OXA-23 carbapenemase in China.

Epidemiology and antimicrobial susceptibility profiles of Gram-negative bacteria causing urinary tract infections in the Asia-Pacific region: 2009-2010 results from the Study for Monitoring Antimicrobial Resistance Trends (SMART)

In 2009, the Study for Monitoring Antimicrobial Resistance Trends (SMART) was expanded to include surveillance of Gram-negative pathogens causing urinary tract infections (UTIs) in the Asia-Pacific region. A total of 1762 isolates were collected from 38 centers in 11 countries from patients with UTIs in 2009 and 2010. In vitro susceptibilities were determined by the broth microdilution method and susceptibility profiles were determined using minimum inhibitory concentration (MIC) interpretive criteria, as recommended by the Clinical and Laboratory Standards Institute (CLSI) in 2010 (M100-S20), in 2011 (M100-S21), and in 2012 (M100-S22). Enterobacteriaceae comprised 86.0% of the isolates, of which Escherichia coli (56.5%) and Klebsiella pneumoniae (13.8%) were the two most common species. Amikacin was the most effective antibiotic (91.7%), followed by ertapenem (86.9%), imipenem (86.6%), and piperacillin-tazobactam (84.9%). Rates of susceptibility were 50.3% for cefoxitin and ranged from 50.3% to 74.2% for the third- and fourth-generation cephalosporins. For ciprofloxacin and levofloxacin, the susceptibility rates were 51.4% and 54.4%, respectively. Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae comprised 28.2% of all isolates. We also found a high rate of resistance to carbapenems among Acinetobacter baumannii and Pseudomonas aeruginosa causing UTI. Interestingly, according to 2012 CLSI breakpoints, approximately 33.4% of ESBL producers were still susceptible to ceftazidime. However, this in vitro efficacy of ceftazidime needs to be validated in vivo by clinical data. The lowered CLSI interpretive breakpoints for piperacillin-tazobactam, carbapenems, and some cephalosporins in 2011-2012 for Enterobacteriaceae resulted in an approximate 5% drop in susceptibility rates for each drug, with the exception of imipenem for which the susceptibility rate dropped from 99.4% according to 2010 criteria to 91.2% according to 2011 criteria. With the updated CLSI criteria, the antimicrobial resistance threat from UTI pathogens in the Asia Pacific area was revealed to be more prominent.

Characterization of Community Acquired Staphylococcus aureus Associated with Skin and Soft Tissue Infection in Beijing: High Prevalence of PVL+ ST398

Adult community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-susceptible S aureus (CA-MSSA) skin and soft tissue infection (SSTI) in China is not well described. A prospective cohort of adults with SSTI was established between January 2009 and August 2010 at 4 hospitals in Beijing. Susceptibility testing and molecular typing, including multilocus sequence typing, spa, agr typing, and toxin detection were assessed for all S. aureus isolates. Overall, 501 SSTI patients were enrolled. Cutaneous abscess (40.7%) was the most common infection, followed by impetigo (6.8%) and cellulitis (4.8%). S. aureus accounted for 32.7% (164/501) of SSTIs. Five isolates (5/164, 3.0%) were CA-MRSA. The most dominant ST in CA-MSSA was ST398 (17.6%). The prevalence of Panton-Valentine Leukocidin (pvl) gene was 41.5% (66/159) in MSSA. Female, younger patients and infections requiring incision or drainage were more commonly associated with pvl-positive S. aureus (P<0.03); sec gene was more often identified in CC5 (P<0.03); seh gene was more prevalent in CC1 (P = 0.001). Importantly, ST59 isolates showed more resistance to erythromycin, clindamycin and tetracycline, and needed more surgical intervention. In conclusion, CA-MRSA infections were rare among adult SSTI patients in Beijing. Six major MSSA clones were identified and associated with unique antimicrobial susceptibility, toxin profiles, and agr types. A high prevalence of livestock ST398 clone (17.1% of all S. aureus infections) was found with no apparent association to animal contact.

Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014

With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies.

In Vitro Susceptibilities of Yeast Species to Fluconazole and Voriconazole as Determined by the 2010 National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study

ABSTRACT We conducted active, laboratory-based surveillance for isolates from patients with invasive infections across China from August 2009 to July 2010. DNA sequencing methods were used to define species, and susceptibility to fluconazole and voriconazole was determined by the Clinical and Laboratory Standards Institute M44-A2 disk diffusion method but using up-to-date clinical breakpoints or epidemiological cutoff values. Candida spp. made up 90.5% of the 814 yeast strains isolated, followed by Cryptococcus neoformans (7.7%) and other non-Candida yeast strains (1.7%). Bloodstream isolates made up 42.9% of the strains, isolates from ascitic fluid made up 22.1%, but pus/tissue specimens yielded yeast strains in <5% of the cases. Among the Candida isolates, Candida albicans was the most common species from specimens other than blood (50.1%) but made up only 23% of the bloodstream isolates (P < 0.001). C. parapsilosis complex species were the most common Candida isolates from blood (33.2%). Uncommon bloodstream yeast strains included Trichosporon spp., C. pelliculosa, and the novel species C. quercitrusa, reported for the first time as a cause of candidemia. Most (>94%) of the isolates of C. albicans, C. tropicalis, and the C. parapsilosis complex were susceptible to fluconazole and voriconazole, as were all of the Trichosporon strains; however, 12.2% of the C. glabrata sensu stricto isolates were fluconazole resistant and 17.8% had non-wild-type susceptibility to voriconazole. Seven C. tropicalis strains were cross-resistant to fluconazole and voriconazole; six were from patients in the same institution. Resistance to fluconazole and voriconazole was seen in 31.9% and 13.3% of the uncommon Candida and non-Candida yeast strains, respectively. Causative species and azole susceptibility varied with the geographic region. This study provided clinically useful data on yeast strains and their antifungal susceptibilities in China.

Phenotypic and genotypic characterization of Enterobacteriaceae with decreased susceptibility to carbapenems: results from large hospital-based surveillance studies in China.

ABSTRACT The resistance mechanism of 49 Enterobacteriaceae isolates with decreased susceptibility to carbapenems collected from 2004 to 2008 at 16 teaching hospitals in China was investigated. Moderate- to high-level carbapenem resistance in most isolates was more closely associated with loss or decreased expression of both major porins combined with production of AmpC or extended-spectrum β-lactamase enzymes, while KPC-2, IMP-4, and IMP-8 carbapenemase production may lead to a low to moderate level of carbapenem resistance in Enterobacteriaceae in China.

Antimicrobial resistance trends among 5608 clinical Gram-positive isolates in China: results from the Gram-Positive Cocci Resistance Surveillance program (2005-2010)

A total of 5608 clinical isolates of Gram-positive bacteria were collected from 12 teaching hospitals across China from 2005 to 2010. The minimum inhibitory concentrations (MICs) of 19 antimicrobial agents were determined by the agar dilution method at the central laboratory. Overall, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRSCoN) were 46.8% and 81.5%, respectively. Isolates from inpatients exhibited a higher rate of MRSA than that from outpatients (52.3% versus 26.2%, P < 0.001). The prevalence of MRSA in respiratory infections (67.5%) was higher than in other sources of infections (P < 0.001). A shift in vancomycin MICs from <0.5 to 1.0 μg/mL was observed during the 6-year period. In 2005, 70.5% of S. aureus isolates were inhibited at the vancomycin MIC of 0.5 μg/mL, while in 2010, 89% of the isolates were inhibited at the vancomycin MIC of 1 μg/mL. With the use of penicillin oral breakpoints, penicillin-resistant Streptococcus pneumoniae (PRSP) increased from 28.6% in 2005 to 59.5% in 2010 and varied among different age groups, with an average rate of 70.6% for children under 5 years old. Importantly, an obvious penicillin MIC right shift was observed from 0.032 to 4 μg/mL during the study period. Serotyping for the isolates from 2005 and 2010 indicated that the high rate of PRSP could be due to the increased prevalence of serogroup 19. The prevalence of vancomycin-resistant enterococci (VRE) increased from 0 in 2005 to 4.9% in 2010. Of the 27 VRE isolates, vanA gene was the most prevalent gene. During the study period, 97.9-100% of different species tested were susceptible to teicoplanin. Linezolid and tigecycline showed potent activities, and no resistant isolate was identified. In conclusion, although the prevalence of MRSA and MRSCoN remained stable over the 6 years, a sharp increase in the prevalence of PRSP was identified. In addition, MIC shifts, including the MICs of penicillin against S. pneumoniae and vancomycin against S. aureus, were observed. Continuous surveillance is warranted to evaluate the resistance trend of clinically important Gram-positive organisms in the future.

In vitro activity of ceftobiprole, linezolid, tigecycline, and 23 other antimicrobial agents against Staphylococcus aureus isolates in China

We investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in China and determined the susceptibility of S. aureus to 26 antimicrobial agents, including ceftobiprole, linezolid, and tigecycline. A total of 798 isolates were collected and tested by agar dilution. The mean prevalence of MRSA was 50.4%, the highest in Shanghai (80.3%), followed by those in Beijing (55.5%) and Shenyang (50.0%). Only 4.2% to 12.6% of MRSA were susceptible to erythromycin, fluoroquinolones, gentamicin, and tetracycline. All isolates were susceptible to teicoplanin, vancomycin, linezolid, tigecycline, and ceftobiprole.

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

ABSTRACT Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.

Antifungal susceptibilities of Candida glabrata species complex, Candida krusei, Candida parapsilosis species complex and Candida tropicalis causing invasive candidiasis in China: 3 year national surveillance

OBJECTIVES To define the antifungal susceptibility patterns of the most common non-albicans Candida spp. in China. METHODS We evaluated the susceptibilities to nine antifungal drugs of Candida parapsilosis species complex, Candida tropicalis, Candida glabrata species complex and Candida krusei isolates from patients with invasive candidiasis at 11 hospitals over 3 years. Isolates were identified by MALDI-TOF MS supplemented by DNA sequencing. MICs were determined by Sensititre YeastOne(TM) using current clinical breakpoints/epidemiological cut-off values to assign susceptibility (or WT), and by CLSI M44-A2 disc diffusion for fluconazole and voriconazole. RESULTS Of 1072 isolates, 392 (36.6%) were C. parapsilosis species complex. C. tropicalis, C. glabrata species complex and C. krusei comprised 35.4%, 24.3% and 3.7% of the isolates, respectively. Over 99.3% of the isolates were of WT phenotype to amphotericin B and 5-flucytosine. Susceptibility/WT rates to azoles among C. parapsilosis species complex were ≥97.5%. However, 11.6% and 9.5% of C. tropicalis isolates were non-susceptible to fluconazole and voriconazole, respectively (7.1% were resistant to both). Approximately 14.3% of C. glabrata sensu stricto isolates (n = 258) were fluconazole resistant, and 11.6% of C. glabrata sensu stricto isolates were cross-resistant to fluconazole and voriconazole. All C. krusei isolates were susceptible/WT to voriconazole, posaconazole and itraconazole. Overall, 97.7%-100% of isolates were susceptible to caspofungin, micafungin and anidulafungin, but 2.3% of C. glabrata were non-susceptible to anidulafungin. There was no azole/echinocandin co-resistance. Disc diffusion and Sensititre YeastOne(TM) methods showed >95% categorical agreement for fluconazole and voriconazole. CONCLUSIONS In summary, reduced azole susceptibility was seen among C. tropicalis. Resistance to echinocandins was uncommon.