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What is known about this topic? Coronavirus disease 2019 (COVID-19) is widespread globally. In China, COVID-19 has been well controlled and has appeared only in importation-related cases. Local epidemics occur sporadically in China and have been contained relatively quickly. What is added by this report? Epidemiological investigation with genome sequence traceability analysis showed that the first case of COVID-19 in Nangong City acquired infection from a confirmed case from Shijiazhuang City; infection subsequently led to 76 local cases. All cases were associated with the index case, and most were located in Fenggong Street and did not spread outside of Nangong City. The main routes of transmission were family clusters, intra-unit transmission, and nosocomial transmission. What are the implications for public health practice? This study highlights new techniques for rapidly tracing cases and identifying COVID-19 transmission chains. The different epidemiological characteristics in Nangong City, from the earliest stages of the outbreak, suggest that allocation of health sources for prevention and treatment were reasonable. Preventing transmission within medical institutions and isolation facilities and strengthening management in the community should be priorities for COVID-19 control during a city lockdown.

Background It has been performed worldwidely to explore the potential of animals that might be a reservoir for community associated human infections of Clostridioides difficile. Several genetically undistinguished PCR ribotypes of C. difficile from animals and human have been reported, illustrating potential transmission of C. difficile between them. Pig and calf were considered as the main origins of C. difficile with predominant RT078 and RT033, respectively. As more investigations involved, great diversity of molecular types from pig and calf were reported in Europe, North American and Australia. However, there were quite limited research on C. difficile isolates from meat animals in China, leading to non-comprehensive understanding of molecular epidemiology of C. difficile in China. Results A total of 55 C. difficile were isolated from 953 animal stool samples, within which 51 strains were from newborn dairy calf less than 7 days in Shandong Province. These isolates were divided into 3 STs and 6 RTs, of which ST11/RT126 was predominant type, and responsible for majority antibiotic resistance isolates. All the isolates were resistant to at least one tested antibiotics, however, only two multidrug resistant (MDR) isolates were identified. Furthermore, erythromycin (ERY) and clindamycin (CLI) were the two main resistant antibiotics. None of the isolates were resistant to vancomycin (VAN), metronidazole (MTZ), tetracycline (TET), and rifampin (RIF). Conclusions In this study, we analyzed the prevalence, molecular characters and antibiotic resistance of C. difficile from calf, sheep, chicken, and pig in China. Some unique features were found here: first, RT126 not RT078 were the dominant type from baby calf, and none isolates were got from pig; second, on the whole, isolates from animals display relative lower resistant rate to these 11 tested antibiotics, compared with isolates from human in China in our previous report. Our study helps to deep understanding the situation of C. difficile from economic animals in China, and to further study the potential transmission of C. difficile between meat animals and human.

An outbreak of coronavirus disease 2019 (COVID-19) of Omicron BA.2 emerged in Jilin City since March 3, 2022, which involved in 27,036 cases by April 12. The vaccination program with inactivated COVID-19 vaccines has been implemented since the beginning of 2021.The incidences of moderate, severe, and critical cases in the whole population of the group of 0+1 dose were 1.82-, 9.49-, and 3.85-fold higher than those in the group of 2 doses, and 5.03-, 44.47-, and ∞-fold higher than those received 3 doses vaccination. For the population ≥60 years, the incidences of moderate, severe, and critical cases in the group of 0+1 dose were 29.92, 9.62, and 4.27 per 100,000, showing 4.13-, 43.72-, and 4.85-fold higher than 2 doses, as well as 13.28-, 22.37-, and ∞-fold higher than 3 doses.The incidences of each type of COVID-19 in the population who were fully vaccinated or booster vaccinated in Jilin City were significantly lower than those who were unvaccinated and/or partially vaccinated. Booster vaccination with homologous inactivated vaccines induces stronger protectiveness for COVID-19 caused by variant of concern (VOC) Omicron.

Summary The study is to determine the effects of milk powder on bone density and metabolism in healthy adolescents. Vitamin D and calcium supplements increased IGF-1 but did not affect bone mineralization or turnover. Higher vitamin D in combination with sufficient calcium supplementation in such populations requires attention. Introduction Both calcium and vitamin D play an important role in bone mineralization in adolescents. Methods In this one and a half-year randomized controlled trial, 232 participants (aged 12–15 years) were randomly assigned to three intervention groups receiving milk powder fortified with vitamin D 400 IU plus calcium 300, or 600, or 900 mg [Ca3D (n = 54), Ca6D (n = 56), and Ca9D (n = 49)], or one control group maintaining habitual diet [control (n = 73)]. Bone turnover markers, serum intact PTH, 25(OH)D, and IGF-1 levels were measured at baseline and one and a half years, and bone mineral contents and bone areal mineral density were measured by Dual-energy X-ray absorptiometry (DXA) at baseline, one year, and one and a half years. Results Baseline average serum 25(OH)D level and calcium intake were 29.4 nmol/L and 363.5 mg/day, respectively. There was a significant increase in bone turnover, total body, hip, lumbar spine bone mineral density (BMD), and total body BMC as well as slight fluctuations in 25(OH)D levels over one and a half years without between-group differences. Significantly decreased serum PTH level was only observed in the Ca6D group (31%, p

BackgroundThe second wave of coronavirus disease 2019 (COVID-19) has been incessantly causing catastrophe worldwide, and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants causes further uncertainty regarding epidemic risk. Here, a novel strategy for the detection of SARS-CoV-2 variants using multiplex PCR coupled with MALDI-TOF MS was developed.MethodsPlasmids carrying gene sequences containing 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants were synthesized. Using the nucleic acid sequence of SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2-variant-carrying plasmid, a MALDI-TOF MS method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect the above nine mutation types. The detection limit of this method was determined via the concentration gradient method. Twenty-one respiratory tract pathogens (9 bacteria, 11 respiratory viruses) and pharyngeal swab nucleic acid samples from healthy people were selected for specific validation. Sixteen samples from COVID-19 patients were used to verify the accuracy of this method.ResultsThe 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and all six variants (B.1.1.7, B.1.351, B.1.429, B.1.526, P.1 and B.1.617) could be identified. The detection limit for all 9 sites was 1.5×103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR CT values less than 30 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge.ConclusionsMultiplex PCR-MALDI-TOF MS provides a new detection option with practical application value for SARS-CoV-2 and its variant infection.Key pointAn all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-MALDI-TOF MS system was developed. All of the SARS-CoV-2 variants can be identified based on 9 types of 7 mutated sites of RBD of spike protein using this method.

Yue Jin, Jie Xue, Mengran Ruan, Jinxing Lu, Qian Xu, Yuanyuan Shi, Fei Yu Department of Pediatrics, Affiliated Shuyang Hospital of Xuzhou Medical University, Shuyang, 223600, Jiangsu, People’s Republic of ChinaCorrespondence: Yue JinDepartment of Pediatrics, Affiliated Shuyang Hospital of Xuzhou Medical University, No. 9, Yingbin Road, Shucheng Town, Shuyang, 223600, Jiangsu, People’s Republic of ChinaTel +86-14762621960Fax +86-527-80817088Email yuejin100@163.comObjective: To investigate the expression of serum miR-155 in children with Mycoplasma pneumoniae pneumonia (MPP).Methods: A total of 100 children at our hospital with pneumonia caused by Mycoplasma pneumoniae infection were enrolled as a study group, including 45 cases in the acute phase (acute phase group) and 55 in the recovery phase (recovery phase group). An additional 30 healthy children were enrolled during the same period as the control group. The expression levels of miR-155, tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), IL-10, IL-13, immunoglobulin (Ig) G, IgA, complements (C3 and CH50), and T lymphocyte subsets (CD3+, CD4+, CD8+, and CD4+/CD8+) were determined. Multivariate logistic regression analysis was performed to identify risk factors affecting MPP in children.Results: miR-155, IL-10, IgG, IgA, CD3+, CD4+, and CD4+/CD8+ were poorly expressed in children with MPP, and their expression in the acute phase group was significantly lower than that in the recovery phase group. TNF-α, IL-13, C3, and CH50 were highly expressed in the children, and their expression was significantly higher in the acute phase group than in the recovery phase group. In the acute phase group, the expression of IL-8 was significantly higher than that in the control and recovery phase groups but without any significant differences between the recovery phase and control groups. Age, season, low complement state, epidemiological contact history, and antibiotic use time were independent risk factors affecting MPP in children.Conclusion: Serum miR-155 is poorly expressed in children with MPP, and it can regulate inflammatory disorders and immune responses.Keywords: miR-155, Mycoplasma pneumoniae, immunity, inflammation, risk factors

In the foaming ceramic production line, the AGV is used as the conveying unit, which is responsible for conveying the kiln tools needed for production from the kiln tail roller to the kiln head roller. Through the AGV car related sensors data, with the aid of Plant Simulation Simulation software and SQL Server 2017 database connection, to modify the Simulation model, Simulation validate the rationality of the AGVS path planning and AGVS system parameter Settings of the optimization results, provide a plan to build the Simulation model based on real-time data, effectively improve the integrity of the model, accurate to improve the accuracy of the model, Simulation and data analysis on the actual production model has significant guiding role.

World Standards Day was established by the International Organization for Standardization (ISO) on October 14 to enhance awareness on the significance of international standardization to respond to the needs of businesses, industries, governments and consumers worldwide Since its rejoining the ISO in 1978, China held various presentations, symposiums, and commemorative events for World Standards Day in major and medium-sized cities nationwide to publicize the role of standardization in societal development and enhance the awareness of standardization among the people The theme for the 51st World Standards Day in this year is “Protecting the planet with standards” Since the coronavirus disease 2019 (COVID-19) pandemic emerged, practitioners in standardization took immediate actions to respond China’s State Standardization Administration, National Health Commission, local governments, and social organizations took emergency actions to formulate and publish a series of relevant national standards, industry standards, local standards, and organization standards for the prevention and control of COVID-19, which played an essential role in regulating and directing efforts in the prevention and control of the outbreak and in protecting the people’s health and safety

Objective To investigate the expression of serum miR-155 in children with Mycoplasma pneumoniae pneumonia (MPP). Methods A total of 100 children at our hospital with pneumonia caused by Mycoplasma pneumoniae infection were enrolled as a study group, including 45 cases in the acute phase (acute phase group) and 55 in the recovery phase (recovery phase group). An additional 30 healthy children were enrolled during the same period as the control group. The expression levels of miR-155, tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), IL-10, IL-13, immunoglobulin (Ig) G, IgA, complements (C3 and CH50), and T lymphocyte subsets (CD3+, CD4+, CD8+, and CD4+/CD8+) were determined. Multivariate logistic regression analysis was performed to identify risk factors affecting MPP in children. Results miR-155, IL-10, IgG, IgA, CD3+, CD4+, and CD4+/CD8+ were poorly expressed in children with MPP, and their expression in the acute phase group was significantly lower than that in the recovery phase group. TNF-α, IL-13, C3, and CH50 were highly expressed in the children, and their expression was significantly higher in the acute phase group than in the recovery phase group. In the acute phase group, the expression of IL-8 was significantly higher than that in the control and recovery phase groups but without any significant differences between the recovery phase and control groups. Age, season, low complement state, epidemiological contact history, and antibiotic use time were independent risk factors affecting MPP in children. Conclusion Serum miR-155 is poorly expressed in children with MPP, and it can regulate inflammatory disorders and immune responses.

BACKGROUNDThe top priority for the control of COVID-19 pandemic currently is the development of a vaccine. A phase 2 trial conducted to further evaluate the immunogenicity and safety of a SARS-CoV-2 inactivated vaccine (CoronaVac).METHODSWe conducted a randomized, double-blind, placebo-controlled trial to evaluate the optimal dose, immunogenicity and safety of the CoronaVac. A total of 600 healthy adults aged 18-59 years were randomly assigned to receive 2 injections of the trial vaccine at a dose of 3 μg/0.5 mL or 6 μg /0.5mL, or placebo on Day 0,14 schedule or Day 0,28 schedule. For safety evaluation, solicited and unsolicited adverse events were collected after each vaccination within 7 days and 28 days, respectively. Blood samples were taken for antibody assay.RESULTSCoronaVac was well tolerated, and no dose-related safety concerns were observed. Most of the adverse reactions fell in the solicited category and were mild in severity. Pain at injection site was the most frequently reported symptoms. No Grade 3 adverse reaction or vaccine related SAEs were reported. CoronaVac showed good immunogenicity with the lower 3 μg dose eliciting 92.4% seroconversion under Day 0,14 schedule and 97.4% under Day 0,28 schedule. 28 days after two-dose vaccination, the Nab levels of individual schedules range from 23.8 to 65.4 among different dosage and vaccination schedules.CONCLUSIONSFavorable safety and immunogenicity of CoronaVac was demonstrated on both schedules and both dosages, which support the conduction of phase 3 trial with optimum schedule/dosage per different scenarios.

Vaccine candidate tested in monkeys Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an urgent race to develop a vaccine. Gao et al. report preclinical results of an early vaccine candidate called PiCoVacc, which protected rhesus macaque monkeys against SARS-CoV-2 infection when analyzed in short-term studies. The researchers obtained multiple SARS-CoV-2 strains from 11 hospitalized patients across the world and then chemically inactivated the harmful properties of the virus. Animals were immunized with one of two vaccine doses and then inoculated with SARS-CoV-2. Those that received the lowest dose showed signs of controlling the infection, and those receiving the highest dose appeared more protected and did not have detectable viral loads in the pharynx or lungs at 7 days after infection. The next steps will be testing for safety and efficacy in humans. Science , this issue p. 77

The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3μg or 6 μg per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans.One Sentence SummaryA purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

A rapid and accurate method to identify the species and antibiotic resistance of Candida spp. in blood is vital to increase the survival rates of patients with bloodstream infections. However, the extremely low levels of Candida spp. in blood make rapid diagnosis by standard blood culture difficult. In this study, we constructed a direct blood culturing method (i.e., the M1 method) by a rapid enrichment method with magnetic beads coated with a recombined human mannan-binding lectin (rhMBL; i.e., M1 protein), which demonstrated much higher Candida sp.-binding capacity than that of full-length MBL expressed in vitro (i.e., M2). With the M1 method, individual colonies were obtained before the standard blood culture method for each species of Candida spp. tested at

To identify the prevalence and characteristics of community-acquired Clostridium difficile infection (CA-CDI) in southwest China, we conducted a cross-sectional study. 978 diarrhea patients were enrolled and stool specimens’ DNA was screened for virulence genes. Bacterial culture was performed and isolates were characterized by PCR ribotyping and multilocus sequence typing. Toxin genes tcdA and/or tcdB were found in 138/978 (14.11%) cases for fecal samples. A total of 55 C. difficile strains were isolated (5.62%). The positive rate of toxin genes and isolation results had no statistical significance between children and adults groups. However, some clinical features, such as fecal property, diarrhea times before hospital treatment shown difference between two groups. The watery stool was more likely found in children, while the blood stool for adults; most of children cases diarrhea ≤3 times before hospital treatment, and adults diarrhea >3 times. Independent risk factor associated with CA-CDI was patients with fever. ST35/RT046 (18.18%), ST54/RT012 (14.55%), ST3/RT001 (14.55%) and ST3/RT009 (12.73%) were the most distributed genotype profiles. ST35/RT046, ST3/RT001 and ST3/RT009 were the commonly found in children patients but ST54/RT012 for adults. The prevalence of CA-CDI in Yunnan province was relatively high, and isolates displayed heterogeneity between children and adults groups.

// Min Yuan 1 , Hai Chen 2 , Xiong Zhu 2 , Jiao Feng 3 , Zhe Zhan 3 , Defu Zhang 3 , Xia Chen 1 , Xiaofei Zhao 1 , Jinxing Lu 1 , Jianguo Xu 1 , Dongsheng Zhou 3 and Juan Li 1 1 State Key Laboratory of Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China 2 Department of Clinical Laboratory, People’s Hospital of Sanya, Hainan 572000, China 3 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Correspondence to: Juan Li, email: lijuan@icdc.cn Dongsheng Zhou, email: dongshengzhou1977@gmail.com Keywords: Pseudomonas putida , multidrug resistant, IMP-45, armA, pSY153-MDR Received: May 07, 2017 Accepted: June 30, 2017 Published: July 22, 2017 ABSTRACT This work characterized mega plasmid pSY153-MDR, carrying bla IMP-45 and armA , from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. bla IMP-45 and armA coexisted in a Tn 1403 –Tn 1548 region, which was generated from homologous recombination of Tn 1403 - and Tn 1548 -like transposons. The second copy of armA was a component of the IS CR28 – armA –∆IS CR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.

Additional file 1. The gross anatomy of the abdominal cavities between infected and control tree shrew. A. Normal control; B. infected animal.

Additional file 2. COG and KEGG pathway annotation results based on PICRUSt.

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

Mobile genetic elements play a key role in the continuing evolution of Clostridium difficile, resulting in the emergence of new phenotypes for individual isolates. On the basis of whole-genome sequencing analysis, we comprehensively explored transposons, CRISPR, prophage, and genetic sites for drug resistance within clade 4 C. difficile isolates with different sequence types. Great diversity in MGEs and a high rate of multidrug resistance were found within this clade, including new transposons, Tn4453a/b with aac(6′) aph(2′′) instead of catD, and a relatively high rate of prophage-carried CRISPR arrays. These findings provide important new insights into the mechanism of genome remodeling within clade 4 and offer a new method for typing and tracing the origins of closely related isolates., Horizontal gene transfer of mobile genetic elements (MGEs) accounts for the mosaic genome of Clostridium difficile, leading to acquisition of new phenotypes, including drug resistance and reconstruction of the genomes. MGEs were analyzed according to the whole-genome sequences of 37 C. difficile isolates with a variety of sequence types (STs) within clade 4 from China. Great diversity was found in each transposon even within isolates with the same ST. Two novel transposons were identified in isolates ZR9 and ZR18, of which approximately one third to half of the genes showed heterogenous origins compared with the usual intestinal bacterial genes. Most importantly, catD, known to be harbored by Tn4453a/b, was replaced by aac(6′) aph(2′′) in isolates 2, 7, and 28. This phenomenon illustrated the frequent occurrence of gene exchanges between C. difficile and other enterobacteria with individual heterogeneity. Numerous prophages and CRISPR arrays were identified in C. difficile isolates of clade 4. Approximately 20% of spacers were located in prophage-carried CRISPR arrays, providing a new method for typing and tracing the origins of closely related isolates, as well as in-depth studies of the mechanism underlying genome remodeling. The rates of drug resistance were obviously higher than those reported previously around the world, although all isolates retained high sensitivity to vancomycin and metronidazole. The increasing number of C. difficile isolates resistant to all antibiotics tested here suggests the ease with which resistance is acquired in vivo. This study gives insights into the genetic mechanism of microevolution within clade 4. IMPORTANCE Mobile genetic elements play a key role in the continuing evolution of Clostridium difficile, resulting in the emergence of new phenotypes for individual isolates. On the basis of whole-genome sequencing analysis, we comprehensively explored transposons, CRISPR, prophage, and genetic sites for drug resistance within clade 4 C. difficile isolates with different sequence types. Great diversity in MGEs and a high rate of multidrug resistance were found within this clade, including new transposons, Tn4453a/b with aac(6′) aph(2′′) instead of catD, and a relatively high rate of prophage-carried CRISPR arrays. These findings provide important new insights into the mechanism of genome remodeling within clade 4 and offer a new method for typing and tracing the origins of closely related isolates.

Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with the membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membranes (MIMs), improvement (by 10- to 50-fold) in the limit of quantitation could be achieved, compared to conventional nanoelectrospray ionization methods or other ambient ionization methods. MIMs were prepared by cross-linking a synthesized molecularly imprinted polymer layer onto a polyvinylidene difluoride (PVDF) membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using mass spectroscopy (MS), and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil, and antibiotic residues in milk.

Rationale Antibiotic resistance in pathogenic bacteria is becoming a global public health problem, such as aminoglycoside resistance encoded by the armA gene. Although many methods have been reported, rapid analysis of environmental samples is still challenging. Method A rapid analytical method was developed in this study to determine bacterial aminoglycoside resistance using membrane electrospray ionization mass spectrometry (MESI-MS). Precursor/product-ion pairs of ArmA unique peptides were detected with minimal sample preparation. Standard peptides were synthesized and used for developing and validating the methodology, and then the method was verified by both ArmA positive and ArmA negative simulated environmental samples. Results A rapid method for determination of bacterial aminoglycoside resistance was developed using MESI-MS/MS. The bacterial cultural time was optimized to 2 hours, and the precision, accuracy and recovery of this method were investigated. The peptide IHSSTNER (IR-8) unique to ArmA in simulated environmental samples can be successfully identified within 3 hours. Conclusions The novel assay offered a rapid method to determine bacterial aminoglycoside resistance with high sensitivity, accuracy and precision in simulated environmental samples. This method could also be applied to identify other drug-resistance proteins in clinical/environmental samples. Copyright © 2016 John Wiley & Sons, Ltd.

BackgroundClade 5Clostridioides difficilediverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11C. difficileisolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates.ResultsIsolates 10,010 and 12,038 carried a distinct deletion intcdAcompared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations intcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes.ConclusionsWe concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

Background: Escherichia coli sequence type (ST) ST131 is one of the most widespread and successful opportunistic pathogenic E. coli clones known as multiple-resistance. It can cause community- and hospital-acquired urinary tract infection, abdominal and pelvic infection, and bacteraemia with high morbidity and mortality. Currently, carbapenems have been considered the treatment of choice for E. coli ST131 infections. Although carbapenem resistance is still rare, the resistance rate to carbapenems is growing slowly mainly due to the acquisition of β-lactamase and carbapenemase. In this study, we report a nosocomial outbreak of predominantly respiratory infections caused by blaKPC-2 carrying E. coli ST131 in a Chinese hospital and revealed the transmission of carbapenem-non-susceptible E. coli, as well as the acquisition and spread of blaKPC-2 by E. coli ST131. Methods: A total of 45 carbapenem-non-susceptible E.coli strains were collected in one Beijing hospital from July 2012 to December 2013. The characteristic of these strains including antimicrobial susceptibility, carbapenem resistance mechanism, plasmid profiles and genetic background were determined by two-fold broth microdilution method, PCR, PFGE-S1 southern blot hybridization and whole genome sequencing respectively. Phylogenetic relationships between these strains were inferred. The genetic context of blaKPC-2 was analyzed to trace the spread of blaKPC-2. Findings: 45 carbapenem-non-susceptible E. coli strains were isolated from patients mainly with respiratory infections in this nosocomial outbreak. These strains carried blaKPC-2 and belonged to ST131. These strains are closely related and divided into multiple lineages in the phylogenetic tree. It was initially prevalent only in the ward E02, then spread to other wards and multiple lineages co-evolved, resulting in a nosocomial respiratory infection outbreak of carbapenem-non-susceptible E. coli ST131 in these geriatric wards. The blaKPC-2 positive plasmid profiles suggested that the carbapenem resistance was due to the acquisition of multiple transmissible plasmids carrying blaKPC-2 by E. coli ST131. These plasmids with different backbone sequence exhibit wealthy multidrug resistance (MDR) regions and cointegrate structure at the mediate of IS26. Diverse multidrug resistance elements were transferred and rearranged between these plasmids with different backbone sequence at the mediate of IS26, resulting in the rapid spread of antibiotic-resistant genes and blaKPC-2 between these strains. Interpretation: E. coli ST131 has become a multidrug epidemic clone that can cause hospital-acquired respiratory infections that have been less aware. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections. The emergence of blaKPC in E. coli ST131 may herald the beginning for transmission of blaKPC with high prevalence in communities and hospitals. Close monitoring of blaKPC-2 positive ST131 in hospitals and communities are necessary for control and proper treatment of ST131 infections. Funding Statement: This work was supported by the National Natural Science Foundation of China (81861138053，31761133004 and 81861138004) and the Ministry of Science and Technology (2018ZX10733-402). Declaration of Interests: All authors declare that there are no conflicts of interest. Ethics Approval Statement: Not needed.

Clostridium difficile infection (CDI) has become a worldwide public health problem causing high mortality and a large disease burden. Molecular typing and analysis is key important for surveillance and infection control of CDI. However, molecular characterization of C. difficile across China is extremely rare. To investigate the multicenter data of C. difficile isolates across China, the toxin profile, molecular types, and epidemiological characteristics of C. difficile were analyzed. A total of 199 C. difficile isolates collected from 2010-2015 were analyzed using multilocus sequence typing (MLST), PCR ribotyping, and toxin genes amplification. There were 35 STs and 27 ribotypes (RTs) identified in this study, in which ST35 (15.58%), ST3 (15.08%), ST37 (12.06%) and RT017 (14.07%), RT001 (12.06%), RT012 (11.56%), are the most prevalent types, respectively. One isolate with ST1 and 8 isolates with ST 11 were identified. We identified a new ST in this study, denoted ST332. The toxin profile tcdA+tcdB+tcdC+tcdR+tcdE+CDT- (65.83%) was the predominant profile. Furthermore, 11 isolates with positive binary toxin genes were discovered. According to the PCR ribotyping, ST1 was confirmed as RT 027, and 6 out of the 8 ST11 strains were confirmed as RT 078. The epidemiological characteristics of C. difficile in China shows geographical differences, and both the toxin profile and molecular types exhibit great diversity across the different areas.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that bla NDM-1 was carried on an IncX3 plasmid. The bla IMP-4 and bla KPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12941-017-0231-z) contains supplementary material, which is available to authorized users.

Clostridium difficile is a well-known nosocomial infectious pathogen. Research on C. difficile infection has primarily focused on strains such as the hypervirulent PCR ribotype 027 (sequence type 1 [ST1]) emerging in Europe and North America. However, other new emerging ribotypes in some countries have attracted attention, such as PCR ribotype 17 (ST37) in Asia and Latin America. We collected 70 strains and sequenced their toxin genes, tcdA and tcdB . Multilocus sequence typing (MLST) was used to study their population structure. In addition, tcdA and/or tcdB sequences of 25 other isolates were obtained from GenBank. Single nucleotide polymorphisms (SNPs) were identified and analyzed. Phylogenetic analyses were performed to study toxin gene evolution. All tcdA and tcdB sequences were divided into 1 of 16 types (denoted A01 to -16 and B01 to -16, respectively). Hypervirulent strain RT027 is A13B12, and RT078 is A14B10, whereas the newly epidemic strain RT017 is A15B13. SNP analysis suggests the possibility of recombination in tcdB , perhaps through horizontal gene transfer. SNPs were also found in the sequences corresponding to the PCR primers widely used for toxin detection. Our study shows that ST037 shares a few genotypic features in its tcdA and tcdB genes with some known hypervirulent strains, indicating that they fall into a unique clade. Our findings can be used to map the relationships among C. difficile strains more finely than can be done with less sensitive methods, such as toxinotyping or even MLST, to reveal their inherent epidemiological characteristics.

The biological characteristics and molecular epidemiology of Pseudomonas aeruginosa associated with mink hemorrhagic pneumonia from Shandong province of eastern China were determined in this study. From 2010 to 2011, 30 mink P. aeruginosa isolates were identified from lung, fecal and feed samples of clinical cases and subjected to serotyping, antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) using SpeI. The P. aeruginosa isolates belonged to four serotypes-21 of type G, four of type I, three of type M, one of type B, and one non-typable strain. The strains were divided into four large groups as determined by PFGE. Isolates from the group 2 were highly homologous and were obtained from the same region as an epidemic. All of the isolates were sensitive to piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, amikacin, gentamicin and tobramycin and resistant to ampicillin, cefuroxime and cefuroxime axetil. A high frequency of resistance was found to ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, nitrofurantoin, and trimethoprim/sulfamethoxazole (96.7%). Resistance to ticarcillin/clavulanic acid, ciprofloxacin and levofloxacin was less common (13.3%). There was no relationship between antibiotic resistance and serotype distribution of the isolates. The epidemic serotype of P. aeruginosa from the mink hemorrhagic pneumonia in Shandong province was type G, which was a clone of commonly found in this province. These findings reveal the genetic similarities and antimicrobial susceptibility profiles of P. aeruginosa from clinical cases of mink hemorrhagic pneumonia and will facilitate the prevention and control of the disease in Shandong province of China.

It is known that antibiotic usage is associated with the development of Clostridioides difficile infection (CDI), especially clindamycin, third-generation cephalosporins, and fuoroquinolones. Antibiotic resistance rates to many antibiotics varies a lot by study. We performed a study focused on antibiotic resistance in clinical isolates of C. difficile from more widespread geographic regions across China. Of 319 C. difficile isolates tested against 11 antibiotics, 313 (98.1%) were resistant to at least one antibiotic. The highest rate of resistance was to ciprofloxacin, clindamycin, and erythromycin across all age groups, similar to previous studies. However, all isolates were susceptible to metronidazole and vancomycin. Overall the resistance rate to tested antibiotics was lower than other reports in China except for chloramphenicol and meropenem. Genotype ST37/RT017 in clade 4 was resistant to more antibiotics than other types. Unexpectedly, RT078 isolates in this study were susceptible to almost all tested antibiotics. In addition, the proportion of multi-drug resistant (MDR) isolates observed (17%) in this study was much lower than several European studies (up to 55%) and a previous study in China (78%). Although isolates from patients aged between 65 and 85 were more resistant to antibiotics in comparison to other age groups, MDR isolates were still detected in children below 2-years of age. The highest percentage of MDR isolates was determined in South China, an area that is most developed economically. The clade 4, RT017 (ST37) has been associated with outbreaks in Europe and North America and is responsible for most C. difficile infections (CDIs) in Asia. In addition, RT017 is often clindamycin and fluoroquinolone resistant. This study provided a relatively comprehensive description of antibiotic resistance of C. difficile in China, and further elucidates the epidemiology and antibiotic resistance of clinical isolates of C. difficile in China at a national level.

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.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21–49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

Candida tropicalis is considered as the leading pathogen in nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly in leukemia. The yeast-filament transition is required for virulent infection by Candida. Several studies have explored the genome-wide transcription profile of Candida, however, no report on the transcriptional profile of C. tropicalis under yeast-filament transition has been published. In this study, the transcriptomes of three C. tropicalis isolates with different adhesion and biofilm formation abilities, identified in our previous studies, were analyzed in both the yeast and filament states using RNA-Seq. Differentially expressed genes were found for each isolate during the transition. A total of 115 genes were up- or down- regulated in the two hyphal-producing isolates (ZRCT 4 and ZRCT 45). Among these differentially expressed genes, only two were down-regulated during the yeast-filament transition. Furthermore, six filament-associated genes were up-regulated in the hyphae-producing isolates. According to Candida Hypha Growth Database established in this study, 331 hyphae- related genes were discovered in C. tropicalis. ALS1 and ALS3 were down-regulated and up-regulated, respectively, during filamentous growth of C. tropicalis. These findings proved a better understanding of gene expression dynamics during the yeast-filament transition in C. tropicalis.

Antibiotic resistance in pathogenic bacteria is becoming a global public health problem, such as aminoglycoside resistance encoded by the armA gene. Although many methods have been reported, rapid analysis of environmental samples is still challenging.A rapid analytical method was developed in this study to determine bacterial aminoglycoside resistance using membrane electrospray ionization mass spectrometry (MESI-MS). Precursor/product-ion pairs of ArmA unique peptides were detected with minimal sample preparation. Standard peptides were synthesized and used for developing and validating the methodology, and then the method was verified by both ArmA positive and ArmA negative simulated environmental samples.A rapid method for determination of bacterial aminoglycoside resistance was developed using MESI-MS/MS. The bacterial cultural time was optimized to 2 hours, and the precision, accuracy and recovery of this method were investigated. The peptide IHSSTNER (IR-8) unique to ArmA in simulated environmental samples can be successfully identified within 3 hours.The novel assay offered a rapid method to determine bacterial aminoglycoside resistance with high sensitivity, accuracy and precision in simulated environmental samples. This method could also be applied to identify other drug-resistance proteins in clinical/environmental samples. Copyright © 2016 John WileySons, Ltd.

The prevalence of antimicrobial resistant Klebsiella pneumoniae in poultry products has been a public concern, as it severely endangers food safety and human health. In this study, we investigated 90 antimicrobial resistant Klebsiella strains that were isolated from a commercial broiler slaughter plant in Shandong province of China. Nearly all (89/90) of the isolates were identified as infectious phylogenetic group KpI-type K. pneumoniae. Out of these 90 strains, 87 (96.7%) were multidrug-resistant isolates, and 87 (96.7%) were extended-spectrum beta-lactamase (ESBL)-producing isolates. An analysis of the prevalence of quinolone resistance genes showed that 7.8%, 77.8%, 26.7%, and 2.2% of the strains carried the qnrA, qnrB, qnrS, and qepA genes, respectively. An analysis of beta-lactam resistance genes showed that a high percentage of the strains contain the blaTEM (76.7%), blaSHV (88.9%), and blaCTX-M (75.6%) genes, among which three blaSHV subtypes (blaSHV-1, n = 30; blaSHV-11, n = 38; blaSHV-12, n = 12) and three blaCTX-M subtypes (blaCTX-M-14, n = 14; blaCTX-M-15, n = 35; blaCTX-M-55, n = 19) were found. A further investigation of mobile genetic elements involved in horizontal multidrug resistance gene transfer showed the presence of class 1 and 2 integrons in 77 (85.6%) and five (5.6%) isolates, respectively, while no class 3 integrons were detected. Four types of class 1 integrons containing specific gene cassette arrays (dfrA12–orfF–aadA2, dfrA17–aadA5, dfrA1–aadA1, and empty) were identified. Only one gene cassette array (dfrA1–sat2–aadA1) was detected in the class 2 integrons. Furthermore, four different types of insertion sequence common region 1 (ISCR1)-mediated downstream structures were successfully identified in 46 class 1 integron-positive isolates, among which ISCR1–sapA-like–qnrB2–qacEΔ1 was the most commonly observed structure. Chi-square tests revealed a significant association between ESBL genes, plasmid-mediated quinolone resistance (PMQR) genes, and class 1 integrons (p < 0.01). Additional conjugation experiments confirmed this relationship (p < 0.01) in transconjugants by finding that a high percentage of PMQR genes (74.0%) and class 1 integrons (73.7%) were co-transferred with ESBL genes. Finally, multilocus sequence typing (MLST) was performed, and it revealed that the isolates from chickens are widely distributed in humans, and that antimicrobial resistance is not only disseminated by clonal spreading, but largely by horizontal gene transfer. These results suggest that horizontal transfer of antimicrobial resistance genes by mobile genetic elements, such as integrons, plays a major role in the spread of antimicrobial resistance. Therefore, elucidating the structures of drug resistance integrons is of great importance to the commercial broiler slaughter plant in Shandong, China.