Extensive Genetic Diversity and Epidemiological Patterns of Factor H-Binding Protein Variants among Neisseria meningitidis in China.

Factor H-binding protein (fHbp) is a virulence factor expressed by Neisseria meningitidis (N. meningitidis), the primary causative agent of invasive meningococcal disease (IMD) in humans. fHbp is utilized as the main component in vaccines to provide protection against IMD caused by serogroup B N. meningitidis. In order to comprehensively investigate the genetic diversity and epidemiological patterns of fHbp variants within isolates of Chinese N. meningitidis, we utilized the NEIS0349 locus, which encompasses the complete coding sequences of fHbp. This enabled us to identify allelic variants of fHbp with enhanced resolution. A total of 109 fHbp variants were identified in 1013 Chinese N. meningitidis isolates. We reconstructed a phylogenetic tree and analyzed the epidemiological characteristics of each variant. Considering both temporal and geographical distribution patterns, only four fHbp variants (v2.16, v2.18, v2.404, and v2.21) exhibited persistent nationwide prevalence during the previous decade (2011-2021). These variants were highly prevalent in both serogroup B strains from patients and healthy individuals, suggesting their potential as suitable vaccine candidates for nationwide implementation against IMD caused by serogroup B strains. Our study emphasizes the significance of conducting continuous surveillance of meningococcal strains to monitor the genetic diversity of fHbp for the purpose of vaccine development.

Identification of pneumococcal serotypes with individual recognition of vaccine types by a highly multiplexed real-time PCR-based MeltArray approach.

BACKGROUND: Pneumococcus serotyping is important for monitoring serotype epidemiology, vaccine-induced serotypes replacement and emerging pathogenic serotypes. However, the lack of high-resolution serotyping tools has hindered its widespread implementation. METHODS: We devised a single-step, multiplex real-time polymerase chain reaction (PCR)-based MeltArray approach termed PneumoSero that can identify 92 serotypes with individual recognition of 54 serotypes, including all 24 currently available vaccine types. The limit of detection (LOD) and the ability to coexisting serotypes were studied, followed by analytical evaluation using 92 reference pneumococcal strains and 125 non-pneumococcal strains, and clinical evaluation using 471 pneumococcus isolates and 46 pneumococcus-positive clinical samples. RESULTS: The LODs varied with serotypes from 50 to 100 copies per reaction and 10 % of the minor serotypes were detectable in samples containing two mixed serotypes. Analytical evaluation presented 100 % accuracy in both 92 reference pneumococcal strains and 125 non-pneumococcal strains. Clinical evaluation of 471 pneumococcus isolates displayed full concordance with Sanger sequencing results. The 46 clinical specimens yielded 45 typeable results and one untypeable result. Of the 45 typeable samples, 41 were of a single serotype and four were of mixed serotypes, all of which were confirmed by Sanger sequencing or separate PCR assays. CONCLUSION: We conclude that the PneumoSero assay can be implemented as a routine tool for pneumococcal serotyping in standard microbiology laboratories and even in clinical settings.

Deciphering the pivotal role of people with high-frequency occupational animal exposure in antibiotic resistance transmission between humans and animals.

BACKGROUND: The spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) among humans and food-producing animals has been widely reported. However, the transmission routes and associated risk factors remain incompletely understood. METHODS: Here, we used commensal Escherichia coli bacteria strains from faeces of pigs and local citizens [HEG: high exposure group (pig breeders, butchers or restaurant chefs) and LEG: low exposure group (other occupations)] to explore the dynamics of ARB and ARG transmission between animals and humans. RESULTS: Most ARGs (96%) present in pigs were shared with humans. Carriage rates of the shared ARGs suggest two transmission patterns among pigs, the HEG and LEG: one pattern was highest in pigs, gradually decreasing in the HEG and LEG (e.g. floR and cmlA1); the other pattern was increasing from pigs to the HEG but then decreasing in the LEG (e.g. mcr-1.1). Carriage rates of the HEG were higher than in the LEG in both patterns, implicating the HEG as a crucial medium in transmitting ARB and ARGs between food-producing animals and humans. Moreover, frequent inter/intragroup transmission via strains, plasmids and/or mobile elements was evident. Carriage of mcr-1.1 on human-gut-prevalent plasmids possibly promoted its enrichment in the HEG. CONCLUSIONS: The HEG is a crucial factor in transmitting ARB and ARGs between food-producing animals and humans. Rational measures to contain the risks of occupational exposure are urgently needed to keep dissemination of antibiotic resistance in check and safeguard public health.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction.

Objective: To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes. Methods: A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping. Results: A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains. Conclusion: A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Emergence of blaIMI-2- and blaIMI-16-Producing Enterobacter asburiae in the Aquaculture Environment of Jiangsu, China.

Carbapenem-resistant Enterobacteriaceae strains have emerged as a serious threat to global public health. In recent years, blaIMI, a carbapenemase gene that drew less attention before, has been increasingly detected in both clinical and environmental settings. However, the environmental distribution and transmission of blaIMI, especially in aquaculture, require systematic investigation. In this study, the blaIMI gene was detected in fish (n = 1), sewage (n = 1), river water (n = 1), and aquaculture pond water samples (n = 17) collected from Jiangsu, China, demonstrating a relatively high sample-positive ratio of 12.4% (20/161). Thirteen blaIMI-2- or blaIMI-16-carrying Enterobacter asburiae strains were isolated from blaIMI-positive samples of aquatic products and aquaculture ponds. We also identified a novel transposon (Tn7441) carrying blaIMI-16 and a conserved region containing several truncated insertion sequence (IS) elements harboring blaIMI-2, all of which may play important roles in blaIMI mobilization. The occurrence of blaIMI-carrying Enterobacter asburiae in aquaculture-related water samples and fish samples highlights the risk of transmission of blaIMI-carrying strains through the food chain and the need for effective measures to prevent further dissemination. IMPORTANCE IMI carbapenemases have been detected in clinical isolates of many bacterial species with systemic infection and cause a further burden on clinical treatment in China, but their source and distribution are still unclear. The study systematically investigated the distribution and transmission of the blaIMI gene in aquaculture-related water bodies and aquatic products in Jiangsu Province, China, which is famous for its rich water resources and developed aquaculture industry. The relatively high prevalence of blaIMI in aquaculture samples and the identification of novel mobile elements harboring blaIMI enhance our knowledge of blaIMI gene distribution and highlight the public health risk and urgency of surveillance of aquaculture water systems in China.

A Novel Dual-Emission Fluorescence Probe Based on CDs and Eu3+ Functionalized UiO-66-(COOH)2 Hybrid for Visual Monitoring of Cu2.

In this work, CDs@Eu-UiO-66(COOH)2 (denoted as CDs-F2), a fluorescent material made up of carbon dots (CDs) and a Eu3+ functionalized metal-organic framework, has been designed and prepared via a post-synthetic modification method. The synthesized CDs-F2 presents dual emissions at 410 nm and 615 nm, which can effectively avoid environmental interference. CDs-F2 exhibits outstanding selectivity, great sensitivity, and good anti-interference for ratiometric sensing Cu2+ in water. The linear range is 0-200 µM and the limit of detection is 0.409 µM. Interestingly, the CDs-F2's silicon plate achieves rapid and selective detection of Cu2+. The change in fluorescence color can be observed by the naked eye. These results reveal that the CDs-F2 hybrid can be employed as a simple, rapid, and sensitive fluorescent probe to detect Cu2+. Moreover, the possible sensing mechanism of this dual-emission fluorescent probe is discussed in detail.

A Ratiometric Fluorescent Sensor Based on Dye/Tb (III) Functionalized UiO-66 for Highly Sensitive Detection of TDGA.

Thiodiglycolic acid (TDGA) is a biomarker for monitoring vinyl chloride exposure. Exploring a facile, rapid and precise analysis technology to quantify TDGA is of great significance. In this research, we demonstrate a fluorescent sensor based on dual-emissive UiO-66 for TDGA detection. This ratiometric fluorescent material named C460@Tb-UiO-66-(COOH)2 was designed and synthesized by introducing organic dye 7-diethylamino-4-methylcoumarin (C460) and Tb3+ into UiO-66-(COOH)2. The as-obtained C460@Tb-UiO-66-(COOH)2 samples showed highly selective recognition, excellent anti-interference and rapid response characteristics for the recognition of TDGA. The detection limit is 0.518 mg·mL-1, which is much lower than the threshold of 20 mg·mL-1 for a healthy person. In addition, the mechanism of TDGA-induced fluorescence quenching is discussed in detail. This sensor is expected to detect TDGA content in human urine.

A Novel Turn-On Fluorescence Probe Based on Cu(II) Functionalized Metal-Organic Frameworks for Visual Detection of Uric Acid.

As an important biomarker in urine, the level of uric acid is of importance for human health. In this work, a Cu(II) functionalized metal-organic framework (Cu2+@Tb-MOFs) is designed and developed as a novel fluorescence probe for wide-range uric acid detection in human urine. The study shows that this fluorescence platform demonstrated excellent pH-independent stability, high water tolerance, and good thermal stability. Based on the strong interaction between metal ions and uric acid, the designed Cu2+@Tb-MOFs can be employed as efficient turn-on fluorescent probes for the detection of uric acid with wide detection range (0~104 µM) and high sensitivity (LOD = 0.65 µM). This probe also demonstrates an anti-interference property, as other species coexisted, and the possibility for recycling. The sensing mechanisms are further discussed at length. More importantly, we experimentally constructed a molecular logic gate operation based on this fluorescence probe for intelligent detection of uric acid. These results suggest the Cu(II) functionalized metal-organic framework can act as a prominent candidate for personalized monitoring of the concentration of uric acid in the human urine system.

Characterization of blaKPC-2-Carrying Plasmid pR31-KPC from a Pseudomonas aeruginosa Strain Isolated in China.

This work aimed to characterize a 29-kb blaKPC-2-carrying plasmid, pR31-KPC, from a multidrug resistant strain of Pseudomonas aeruginosa isolated from the sputum of an elderly patient with multiple chronic conditions in China. The backbone of pR31-KPC is closely related to four other blaKPC-2-carrying plasmids, YLH6_p3, p1011-KPC2, p14057A, and pP23-KPC, none of which have been assigned to any of the known incompatibility groups. Two accessory modules, the IS26-blaKPC-2-IS26 unit and IS26-ΔTn6376-IS26 region, separated by a 5.9-kb backbone region, were identified in pR31-KPC, which was also shown to carry the unique resistance marker blaKPC-2. A comparative study of the above five plasmids showed that p1011-KPC2 may be the most complete plasmid of this group to be reported, while pR31-KPC is the smallest plasmid having lost most of its conjugative region. Regions between the iterons and orf207 in the backbone may be hot spots for the acquisition of exogenous resistance entities. The accessory regions of these plasmids have all undergone several biological events when compared with Tn6296. The further transfer of blaKPC-2 in these plasmids may be initiated by either the Tn3 family or IS26-associated transposition or homologous recombination. The data presented here will contribute to a deeper understanding of blaKPC-2 carrying plasmids in Pseudomonas.

A Nosocomial Respiratory Infection Outbreak of Carbapenem-Resistant Escherichia coli ST131 With Multiple Transmissible bla KPC-2 Carrying Plasmids.

Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections, and resistance to carbapenems is emerging due to the acquisition of carbapenemase-encoding genes. In this study, 45 carbapenem-resistant E. coli strains were collected in a hospital. The resistance mechanisms, plasmid profiles, and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of bla KPC-2 was analyzed to trace the potential dissemination of bla KPC-2. The 45 carbapenem-resistant E. coli ST131 strains were closely related. Initially prevalent only in a single ward, ST131 subsequently spread to other ward, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first isolation of E. coli ST131. The bla KPC-2-positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition by E. coli ST131 of transmissible plasmids pE0272_KPC and pE0171_KPC carrying bla KPC-2. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections and the spread of bla KPC -carrying E. coli ST131.

Changes in colistin resistance and mcr-1 abundance in Escherichia coli of animal and human origins following the ban of colistin-positive additives in China: an epidemiological comparative study.

BACKGROUND: Following the discovery and emergence of the plasmid-mediated colistin resistance gene, mcr-1, the Chinese government formally banned colistin as an animal growth promoter on April 30, 2017. Herein, we report patterns in colistin resistance and mcr-1 abundance in Escherichia coli from animals and humans between 2015 and 2019, to evaluate the effects of the colistin withdrawal. METHODS: We did an epidemiology comparative study to investigate: annual production and sales of colistin in agriculture across mainland China according to data from the China Veterinary Drug Association from 2015 to 2018; the prevalence of colistin-resistant E coli (CREC) in pigs and chickens in 23 Chinese provinces and municipalities as reported in the China Surveillance on Antimicrobial Resistance of Animal Origin database from Jan 1, 2015, to Dec 31, 2016, and Jan 1, 2017, to Dec 31, 2018; the presence of residual colistin and mcr-1 in faeces from 118 animal farms (60 pig, 29 chicken, and 29 cattle) across four provinces over July 1, 2017, to August 31, 2017, and July 1, 2018 to August 31, 2018; the prevalence of mcr-1-positive E coli (MCRPEC) carriage in healthy individuals attending routine hospital examinations across 24 provinces and municipalities from June 1 to July 30, 2019, comparing with equivalent 2016 data (June 1 to September 30) from our previous study in the same hospitals; and the patterns in CREC prevalence among hospital E coli infections across 26 provinces and municipalities from Jan 1, 2015, to Dec 31, 2016, and Jan 1, 2018, to Dec 31, 2019, reported on the China Antimicrobial Surveillance Network. FINDINGS: After the ban on colistin as a growth promoter, marked reductions were observed in the production (27 170 tonnes in 2015 vs 2497 tonnes in 2018) and sale (US$71·5 million in 2015 vs US$8·0 million in 2018) of colistin sulfate premix. Across 118 farms in four provinces, mean colistin residue concentration was 191·1 µg/kg (SD 934·1) in 2017 versus 7·5 µg/kg (50·0) in 2018 (p<0·0001), and the median relative abundance of mcr-1 per 16S RNA was 0·0009 [IQR 0·0001-0·0059] in 2017 versus 0·0002 [0·0000-0·0020] in 2018 (p=0·0001). Across 23 provinces and municipalities, CREC was identified in pig faeces in 1153 (34·0%) of 3396 samples in 2015-16 versus 142 (5·1%) of 2781 in 2017-18 (p<0·0001); and in chickens in 474 (18·1%) of 2614 samples in 2015-16 versus 143 (5·0%) of 2887 in 2017-18 (p<0·0001). In hospitals across 24 provincial capital cities and municipalities, human carriage of MCRPEC was identified in 644 (14·3%) of 4498 samples in 2016 versus 357 (6·3%) of 5657 in 2019 (p<0·0001). Clinical CREC infections in 26 provinces and municipalities comprised 1059 (1·7%) of 62 737 E coli infections in 2015-16 versus 794 (1·3%) of 59 385 in 2018-19 (p<0·0001). INTERPRETATION: The colistin withdrawal policy and the decreasing use of colistin in agriculture have had a significant effect on reducing colistin resistance in both animals and humans in China. However, continuous colistin monitoring is essential, in particular to act as an early warning system for colistin stewardship in Chinese hospitals. FUNDING: National Key Research and Development Program of China, National Natural Science Foundation of China, and UK Medical Research Council.

Multiple Cross Displacement Amplification Coupled With Gold Nanoparticles-Based Lateral Flow Biosensor for Detection of the Mobilized Colistin Resistance Gene mcr-1.

Fast dissemination of the mobilized colistin resistance (mcr) gene mcr-1 in Enterobacteriaceae causes a huge threat to the treatment of severe infection. In the current report, a multiple cross displacement amplification (MCDA) coupled with the detection of amplified products by gold nanoparticles-based lateral flow biosensor (LFB) assay (MCDA-LFB) was established to identify the mcr-1 gene with simpleness, rapidity, specificity, and sensitivity. The MCDA-LFB assay was performed at a isothermal temperature (63°C) for only 30 min during the amplification stage, and the reaction products were directly identified by using LFB which obtained the result within 2 min. The entire process of experiments, from templates extraction to result judging, was accomplished in <60 min. For the analytical specificity of this method, all of the 16 mcr-1-producing strains were positive, and all of the non-mcr-1 isolates produced the negative results. The sensitivity of mcr-1-MCDA-LFB assay was as little as 600 fg of plasmid DNA per reaction in pure culture, and approximately 4.5 × 103 CFU/mL (~4.5 CFU/reaction) in spiked fecal samples. Therefore, this technique established in the present study is suitable for the surveillance of mcr-1 gene in clinic and livestock industry.

Gut microbiota community characteristics and disease-related microorganism pattern in a population of healthy Chinese people.

China's population accounts for about 1/5th of the world's total population. Owing to differences in environment, race, living habits, and other factors, the structure of the intestinal flora of Chinese individuals is expected to have unique features; however, this has not been thoroughly examined. Here, we collected faecal samples from healthy adults living in three cities of China and investigated their gut microbiome using metagenomics and bioinformatics technology. We found that 11 core bacterial genera were present in all of the Chinese faecal samples; moreover, several patient characteristics (age, region, body mass index, physical exercise, smoking habits, and alcoholic drink, and yogurt consumption) were found to have different effects on the gut microbiome of healthy Chinese people. We also examined the distribution patterns of disease-related microorganisms (DRMs), revealing which DRMs can potentially be used as markers for assessment of health risk. We also developed a program called "Guthealthy" for evaluating the health status associated with the microbiome and DRM pattern in the faecal samples. The microbiota data obtained in this study will provide a basis for a healthy gut microbiome composition in the Chinese population.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China.

OBJECTIVE: Antimicrobial resistance (AMR) has become a global concern and is especially severe in China. To effectively and reliably provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples. METHODS: A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes. RESULTS: The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction. The sensitivity rate, specificity rate, positive predictive value, negative predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. blaCTX-M, blaTEM, mcr-1, fexA, cfr, optrA, and intI1 showed over 80% prevalence. The dissemination of AMR genes was distinct between the two farms. CONCLUSION: We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Extreme diversity and multiple SCCmec elements in coagulase-negative Staphylococcus found in the Clinic and Community in Beijing, China.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are recognized as a large reservoir of staphylococcal cassette chromosome mec (SCCmec) harboured by Staphylococcus aureus. However, data of SCCmec in CoNS are relatively absent particularly in China. METHODS: Seventy-eight CoNS clinical and 47 community isolates were collected in Beijing. PCR was performed to classify SCCmec types. Under oxacillin treatment, quantitative real-time reverse transcription PCR (qRT-PCR) was performed to compare mecA mRNA levels and mRNA half-life between isolates with single SCCmec element and those with multiple one. Their growth curves were analysed. Their bacterial cell wall integrity was also compared by performing a Gram stain. All ccr complex segments were sequenced and obtained ccr segments were analysed by phylogenetic analyses. RESULTS: All 78 clinical isolates had mecA segments compared with 38% in community isolates (total 47). Only 29% clinical isolates and 33% community isolates (among mecA positive isolates) harboured a single previously identified SCCmec type; notably, 17% clinical isolates and 28% community isolates had multiple SCCmec types. Further studies indicated that isolates with multiple SCCmec elements had more stable mecA mRNA expression compared with isolates with single SCCmec elements. CoNS with multiple SCCmec elements demonstrated superior cell wall integrity. Interestingly, phylogenetic analyses of obtained 70 ccr segments indicated that horizontal gene transfer of the ccr complex might exist among various species of clinical CoNS, community CoNS and S. aureus. CONCLUSIONS: CoNS recovered from patients carried extremely diverse but distinctive SCCmec elements compared with isolates from the community. More attention should be given to CoNS with multiple SCCmec not only because they had superior cell wall integrity, but also because CoNS and S. aureus might acquire multiple SCCmec through the ccr complex.

The Activities of Adhesion and Biofilm Formation by Candida tropicalis Clinical Isolates Display Significant Correlation with Its Multilocus Sequence Typing.

Adhesion and biofilm formation, which can occur on abiotic and biotic surfaces, are key components in Candida pathogenicity. The aims of this study were to infer about the C. tropicalis clinical isolates ability to adhere and form biofilm on abiotic and biotic surfaces and to correlate that with the multilocus sequence typing and other virulence factors. Adhesion and biofilm formation were measured in 68 C. tropicalis isolates from 3 hospitals in China on abiotic (polystyrene) and biotic (human urinary bladder epithelial cell) surfaces by crystal violet assay and 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide reduction assay. In our study, almost all C. tropicalis isolates could adhere and produce biofilm on abiotic and biotic surfaces in a strain-dependent manner. The isolates from blood showed relatively lower adhesion and biofilm capacity on polystyrene surface, but had strong secreted aspartyl proteinase activity. Moreover, significant differences were found among MLST groups for adhesion and biofilm capacity. C. tropicalis in multilocus sequence typing group5 and group6 showed high adhesion and biofilm, while isolates in group1 exhibited low adhesion and biofilm formation. Overall, it is important to note that C. tropicalis isolates adhere to and produce biofilm on abiotic and biotic surfaces with strain specificity. These data will play an important role in subsequent research on the pathogenesis of C. tropicalis.

Detection and dissemination of the colistin resistance gene, mcr-1, from isolates and faecal samples in China.

PURPOSE: A recently identified colistin resistance gene, mcr-1, has been reported in many countries. In this study, we established a new real-time PCR method to detect it. METHODOLOGY: We used a real-time PCR method to detect the mcr-1 gene in a variety of isolates and faecal samples from 20 provinces and municipal cities in China. RESULTS: Of the 2330 isolates (from 10 species) screened, 54 (2.3 %) isolates were positive for mcr-1. All of the mcr-1-positive isolates that were identified belonged to Escherichia coli strains, among which 9, 1, and 44 were identified as enteropathogenic E. coli, enteroadherent E. coli, and non-pathogenic E. coli, respectively. The majority of the mcr-1-positive isolates were obtained from farm animals from eight provinces and municipal cities across China. A total of 337 faecal samples, including 229 human and 108 pet animal faecal samples, were also screened for the mcr-1 gene. Of the 337 samples analyzed, six and eight human and pet animal faecal samples were positive for the mcr-1 gene, respectively. CONCLUSION: The data demonstrate that the mcr-1 gene is highly prevalent in human and animal populations in China. This occurrence suggests that active surveillance of the mcr-1 gene is imperative in curtailing its spread.

Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities.

Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis.

[Study on genotype and virulence of Cryptococcus neoformans and Cryptococcus gattii clinical isolates in Guigang, Guangxi Zhuang Autonomous Region].

OBJECTIVE: To understand the species, genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Guigang, Guangxi Zhuang Autonomous Region. METHODS: A total of 20 Cryptococcus strains were isolated from clinical samples in Guigang from 2009 to 2012. The biological identification was conducted by polymerase chain reaction (PCR) to amplify internal transcribed spacer (ITS) sequences. The serotypes and mating types of C. neoformans and C. gattii were identified by PCR with serotype-specific and mating type-specific primers. The genotype was characterized by PCR fingerprinting and URA5 gene restriction fragment length polymorphism (URA5-RFLP). Phenotype study included growth test at 37 °C, melanin production test and urease test. RESULTS: Among the 20 strains, 19 (95%) were identified as C. neoformans varieties (var.) grubii (serotype A, mating type α, genotype VN I), and only 1 was identified as C. gattii (mating type α, genotype VG I). The results of virulence test showed that all the strains grew well at 37 °C and positive in both urease test and melanin production test. CONCLUSION: C. neoformans var. grubii (serotype A, genotype VN I and mating type α) was the predominant pathogen causing cryptococcosis in Guigang, and C. gattii strain was also detected.

[Study on virulence factors of Candida tropicalis isolated from clinical samples].

OBJECTIVE: To determine the in vitro production of virulence factors for Candida (C.) tropicalis, including aspartyl proteinases, phospholipases and hemolytic activities, describe the regulation of virulence factors varying with time in C. tropicalis, and analyze the differences in aspartyl proteinases and hemolytic activities of C. tropicalis isolated from anatomically distinct sites. METHODS: A total of 64 C. tropicalis strains were spot-inoculated onto bovine albumin agar, egg yolk agar and sheep blood agar plates, respectively. Then the plates were incubated for 24, 48 and 72 hour at 37 °C, respectively. The aspartyl proteinases, phospholipase and hemolytic activities were determined at each time point, respectively. RESULTS: All the C. tropiclais isolates showed positive aspartyl proteinases and hemolytic activities at each time point, but no phospholipases activity was detected in C. tropicalis. On comparison of aspartyl proteinases and hemolytic activities at different time points, aspartyl proteinases activity at 48 and 72 hour was higher than that at 24 hour. During 72 hour, hemolytic activity of C. tropicalis increased. No statistical significant differences in aspartyl proteinases and hemolytic activities of C. tropicalis were observed among different infection sites (P=0.368 and 0.985). CONCLUSION: The C. tropicalis clinical isolates in China have aspartyl proteinases activity, hemolytic activity, but have no phospholipase activity.