Zhixing Chen

Microbiologic study of the pathogens isolated from wound culture among Wenchuan earthquake survivors

On May 12, an unprecedented earthquake struck Wenchuan County, Sichuan, China, and 1823 victims were admitted to West China Hospital, Sichuan, China. A total of 464 nonduplicate clinical isolates from wounds of earthquake victims were analyzed. The results show that the most common pathogen was Staphylococcus aureus, but only 24.4% of the total isolates were Gram-positive bacteria, and 73.2% were Gram-negative bacteria. The isolates were significantly different from isolation pattern of 2004 tsunami in Thailand. The isolation rates of extended-spectrum beta-lactamase producer and pandrug-resistant Acinetobacter baumannii in this study might increase the risk of nosocomial infection. In this situation, clinical microbiologists, infection control staff, and administration decision makers should pay high attention to prevent disaster-associated nosocomial infections.

Spread of imipenem-resistant Acinetobacter baumannii of European clone II in Western China

The aim of this study was to investigate the distribution of resistance genes and the clonal relationship amongst imipenem-resistant Acinetobacter baumannii isolates from ten hospitals in Western China as well as to compare the molecular epidemiological data with those of isolates from two hospitals in Hangzhou and Beijing. Genes encoding OXA carbapenemases, metallo-β-lactamases, AmpC cephalosporinase and carbapenem resistance-associated outer membrane protein (CarO) were screened using polymerase chain reaction (PCR) and sequencing. PCR mapping was performed to determine whether insertion sequence ISAba1 elements preceded OXA carbapenemases and AmpC cephalosporinase. International clonal lineages were identified by sequence type multiplex PCR. Multilocus sequence typing (MLST) was performed to determine the sequence types (STs), and then eBURST algorithm was applied to assign clonal complexes (CCs). In this study, dissemination of acquired ISAba1 preceding the bla(OXA-23-like) gene was the predominant enzymatic resistance mechanism amongst 272 imipenem-resistant isolates. Five isolates harboured the carO gene disrupted by insertion of ISAba1 and three isolates lacked carO. All of the 36 representative isolates belonged to European clone II. Ten STs, including three novel types, were identified. These STs were clustered into CC92 and two distinct singletons. These observations suggest that imipenem-resistant A. baumannii of European clone II, which carries acquired ISAba1 preceding the bla(OXA-23-like) gene and belongs to CC92, has spread within Western China.

Increasing imipenem resistance and dissemination of the ISAba1-associated blaOXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit

The antibiotic susceptibility of Acinetobacter calcoaceticus-Acinetobacter baumannii complex strains recovered from the intensive care unit (ICU) of West China Hospital, Sichuan, PR China, from 2006 to 2009 was investigated. The identification of A. baumannii and analysis of carbapenemase-encoding genes and their relationship with ISAba1 were performed by PCR. Furthermore, a DiversiLab repetitive extragenic palindromic sequence-based PCR (rep-PCR) microbial typing system and a multilocus sequence typing (MLST) scheme were applied to assess the genetic relationship of the isolates. The results showed that the antibiotic susceptibility of the A. calcoaceticus-A. baumannii complex isolates changed and imipenem resistance increased rapidly between 2006 and 2009. The blaOXA-51-like and ISAba1-associated blaOXA-23 genes were prevalent in the imipenem-resistant A. baumannii isolates. However, the blaOXA-58-like gene was found in only one isolate and no metallo-β-lactamase genes were detected. The representative multidrug-resistant A. baumannii isolates were identified as one cluster by rep-PCR fingerprinting and belonged to the clonal complex 92 (CC92) according to MLST. These findings indicate a situation of increasing resistance and wide distribution of class D β-lactamase genes, especially the acquired ISAba1-associated blaOXA-23 gene, in A. baumannii isolates in the ICU of West China Hospital, probably caused by expansion of the CC92 clone.

Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province, China

Previous reports on the molecular characteristics of clinical isolates of Cryptococcus species in China have focused on isolates from southeast China. To obtain a more detailed molecular epidemiology, a total of 92 cryptococcal isolates were collected from Sichuan province. A total of 24 isolates from 12 other provinces were collected for comparative study. Genotypes and mating types of 116 Cryptococcus isolates were determined. Among the 116 isolates, 43 isolates (19 isolates from Sichuan and 24 isolates outside of Sichuan) were analysed by multi‐locus sequence typing (MLST). All 116 clinical isolates were mating type α. Most isolates (114/116) were molecular type VNI and the remaining two isolates were VGI and VGII respectively. MLST results revealed five sequence types (STs) of C. neoformans including two novel STs, with most isolates identified as ST5. The two C. gattii isolates identified in our study were ST44 and ST159. Based on our report and previous studies, there are 15 C. neoformans STs in China which can be divided into three subgroups. The C. gattii isolate from Sichuan could be a scattered subtype of VGII (ST44). Our findings demonstrated that C. neoformans isolates in Sichuan are genetically homogeneous, and ST5 is the epidemic clone of C. neoformans in China.

Enhancing pili assembly and biofilm formation in Acinetobacter baumannii ATCC19606 using non-native acyl-homoserine lactones

BackgroundQuorum Sensing (QS) systems influence biofilm formation, an important virulence factor related to the bacterial survival and antibiotic resistance. In Acinetobacter baumannii, biofilm formation depends on pili biosynthesis, structures assembled via the csuA/BABCDE chaperone-usher secretion system. QS signaling molecules are hypothesized to affect pili formation; however, the mechanism behind this remains unclear. This study aimed to demonstrate the possible role of QS signaling molecules in regulating pili formation and mediating the ability to form biofilms on abiotic surfaces.ResultsReal-time quantitative PCR analysis showed the expression of the csuA/BABCDE genes distinctly increased when co-cultured with C6-HSL (P < 0.05). Under the same experimental conditions, expression of BfmS and BfmR was significantly higher than the control strain (P < 0.05). A subsurface twitching assay showed a switch from a small to a large and structured clone that may result from enhanced twitching motility (P < 0.05). Transmission electron microscopy analysis of cells lifted from a MH broth co-cultured with C6-HSL showed more abundant pili-like structures than the control strain. We then tested the idea that the addition of a QS signal, and therefore induction of chaperone-usher secretion system genes, provides a greater benefit at higher biofilm densities. An assay for the total fluorescence intensity of the biofilm using Confocal Laser Scanning Microscopy revealed an obvious increase.ConclusionOur study demonstrated that, increased transcription of the BfmS and BfmR genes, QS signaling molecules enhance the expression of the chaperone-usher secretion system, and this expression is required for twitching motility in A. baumannii. The concomitant pili expression and strain twitching allowed A. baumannii to attach easily to abiotic surfaces and form biofilms at an earlier timepoint.

Antimicrobial susceptibility of clinical isolates from earthquake victims in Wenchuan

On 12 May 2008, an earthquake measuring 8.0 on the Richter scale struck Wenchuan County, Sichuan, China. Between 12 May and 11 June, 1823 victims were hospitalized in West China Hospital. These patients were severely injured, and most of their wounds were contaminated. Here, the results of bacteriological identification and antibiotic susceptibility testing of 725 non-duplicate isolates from earthquake victims are presented. Gram-negative bacilli were most frequently isolated (71.3%). Only 18.9% of isolates were Gram-positive bacteria; Candida spp. accounted for 9.7%, and Gram-negative cocci for 0.1%. After anaerobic culture, four Clostridium sordellii strains and one Clostridium bifermentans strain were isolated from deep wounds. Specimen culture from earthquake victims revealed a spectrum of pathogens and antibiotic susceptibilities that was different from that usually encountered in West China Hospital, especially concerning methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producers, and multidrug-resistant (MDR) non-fermenting Gram-negative bacilli. The pathophysiology of the injuries in earthquake victims was different from that in the patients who were not earthquake victims. A combination of environmental bacteria with a high proportion of Gram-negative bacteria was often observed in the earthquake victims. Approximately 26% of all earthquake victims were shown to be carriers of MDR microorganisms. Therefore, appropriate microbiological assessment upon admission, and identification of patients to be put in quarantine, is of paramount importance.

The microbiological characteristics of patients with crush syndrome after the Wenchuan earthquake

Abstract To improve the treatment of infectious diseases in patients with crush syndrome, we analyzed the results of bacteriological examinations, including antimicrobial susceptibility patterns, of samples taken from patients with crush syndrome admitted to West China Hospital, Sichuan University, after the Wenchuan earthquake. A total of 210 non-replicate clinical isolates were recovered from 42 of the 66 earthquake victims with crush syndrome. Their mean age was 26.9 ± 15.7 y and 40 of them were male. The length of hospital stay was 14.0 days. Wound, blood, sputa, urine and catheter sample specimens were examined. Gram-negative bacilli, Gram-positive bacteria and fungi accounted for 72.4%, 20.0% and 7.6% of the isolates, respectively. Acinetobacter baumannii and Pseudomonas aeruginosa were the major isolates from wounds. Of the isolated strains, 92.8% occurred at >48 h following admission to the hospital, and most of these agents were common isolates in our hospital. Furthermore, 40.9% of these patients were carriers of multidrug-resistant (MDR) microorganisms. Therefore, we can conclude that patients with crush syndrome may have an increased risk of hospital-acquired infection (HAI), suggesting that it is important to select effective antibiotics to control HAI in a timely manner according to the microbiological data.

Different characteristics of cryptococcal meningitis between HIV-infected and HIV-uninfected patients in the Southwest of China

Cryptococcus is the major pathogen that causes fungal meningitis. In the Peoples Republic of China, especially in the Southwest area, cryptococcal meningitis (CM) in HIV-uninfected patients is more common than in HIV-infected patients. We compared clinical features and laboratory data pertaining to CM in patients with different immunological statuses. Demographic data, clinical manifestations, and laboratory data from inpatients in West China Hospital Sichuan University were collected from June 2009 to June 2014. Patients were grouped according to HIV status. Continuous variables were evaluated by Student t-test or Wilcoxon rank sum tests. Categorical variables were analyzed by χ2 test. Among 85 patients with CM were identified, 53 (62.4%) were HIV-uninfected patients. CM occurred more frequently in males in the HIV-infected group. Compared with HIV-infected patients, HIV-uninfected patients had more leukocytes in their blood and more leukocytes and protein in cerebrospinal fluid. More HIV-uninfected patients had increased cerebrospinal fluid (CSF)/serum albumin ratios, while intrathecal immunoglobulin G (IgG) synthesis was significantly increased. The rate of in-hospital mortality of HIV-infected CM patients was higher. Clinical signs are similar between HIV-uninfected and HIV-infected CM patients. Fewer leukocytes and protein was detected in the CSF and lower local synthesis of IgG in the central nervous system in HIV-infected patients, which reflects their diminished immune response. These characteristics should be noted in order to avoid misdiagnosis. Meningeal enhancement and intrathecal IgG synthesis in the HIV-uninfected group was significantly higher, that may be performance of aggressive inflammatory response and might contribute to a better outcome.

Rapid antimicrobial susceptibility testing by matrix-assisted laser desorption ionization–time of flight mass spectrometry using a qualitative method in Acinetobacter baumannii complex

The transmission and infections of multidrug-resistant bacteria can be prevented by rapid identification and antibiotic susceptibility testing (AST) for pathogenic bacteria in a clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been routinely used as a tool for the identification of pathogens; however, a simple and accurate method for a rapid determination of the antimicrobial susceptibility profile is an urgent requisite. The present study established a method based on mass spectrometry to determine the drug resistance. Acinetobacter baumannii complex isolates were tested as an example. After short-term culture, the isolates were incubated with meropenem of different concentrations to determine the growth or the inhibition of the growth by MALDI-TOF MS. The agreement of minimum inhibitory concentration (MIC) values between MALDI-TOF MS-based rapid AST and broth microdilution method in susceptible and resistant strains was 77.1% and 70.1%, respectively. The susceptibility-breakpoint concentration (2 μg/mL) achieved a 98.9% sensitivity and 100% specificity with respect to resistance detection. Similarly, 96.9% sensitivity and 100% specificity were obtained for resistance detection with meropenem concentration at 8 μg/mL. MALDI-TOF MS-based rapid AST was applied to determine the drug resistance at breakpoint concentration, although MS-MICs might shift to a low dilution. Thus, it is critical for patients to accelerate the AST result from two days to several hours.

Triton X-100 and increased volume of test bacteria in CIM enhanced the detection of carbapenemase-producing Acinetobacter baumannii complex

Acinetobacter baumannii complex has become a public health threat due to its ability to cause outbreaks of infections and acquire resistance to almost all antibiotics, including carbapenem, by producing carbapenemases (1, 2). The Clinical and Laboratory Standards Institute (CLSI) has recently recommended a modified carbapenem inactivation method (mCIM) for the phenotypic detection of carbapenemaseproducing Enterobacteriaceae but not A. baumannii complex (3). Modification of mCIM into a highly effective phenotypic method to detect carbapenemase-producing A. baumannii complex will provide important information to prevent the spread of carbapenem-resistant A. baumannii complex. In this study, 152 clinical isolates of A. baumannii complex from the West China Hospital of Sichuan University, a 4,800-bed tertiary teaching hospital, were included. All strains were previously identified using an automated Vitek 2 Compact instrument (bioMérieux, France) and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) (Bruker Daltonik, GmbH, Bremen, Germany). The strains were tested for carbapenem susceptibility using the Vitek 2 Compact instrument with Vitek 2 ASTGN67 cards and Vitek 2 AST-XN04 cards. The judgment criteria of carbapenem susceptibility for the A. baumannii complex were based on the MICs of doripenem, imipenem, and meropenem according to a CLSI method (4). The PCR assays were performed using a Qiagen M-PCR kit (Qiagen, Germany) to detect the -lactamase genes in all the strains, as previously described (5–7). The PCR products were analyzed by gel electrophoresis (Bio-Rad, USA). Further, we sequenced the gene products with a corresponding visual band. Efflux pump inhibitor MC-207110 (phenylalanine arginyl -naphthylamide [PA N]) (Absin, Shanghai, China) was used for phenotypic screening of efflux pump activity according to a CLSI method (4). Outer membrane protein analysis was performed by the use of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8, 9). The definition for the carbapenemase-positive strains was that they were resistant to carbapenem and harbored the carbapenemase genes, while the definition for the carbapenemasenegative strains was that they were sensitive to carbapenem or were resistant to carbapenem due to porin loss and/or the presence of extended-spectrum -lactamases [ESBLs] coupled with efflux hyperproducers as described previously (10, 11). Of the 152 A. baumannii complex strains, 83 were carbapenemase positive and 69 were carbapenemase negative. Klebsiella pneumoniae strain ATCC BAA-1705 was used as a positive control and Klebsiella pneumoniae ATCC BAA-1706 as the negative control in the phenotypic tests. Accepted manuscript posted online 5 January 2018 Citation Liu M, Song Q, Wu L, Li M, Chen Z, Kang M, Xie Y. 2018. Triton X-100 and increased volume of test bacteria in the carbapenem inactivation method enhanced the detection of carbapenemase-producing Acinetobacter baumannii complex isolates. J Clin Microbiol 56:e01982-17. https://doi.org/10.1128/JCM .01982-17. Editor Nathan A. Ledeboer, Medical College of Wisconsin Copyright