Establishment of a Fast Diagnostic Method for Sepsis Pathogens Based on M1 Bead Enrichment.

Blood culture-based sepsis diagnostic methods usually cannot obtain positive results in a timely manner. Molecular diagnostic methods, such as real-time PCR without blood culture, would be more time-saving and suitable for pathogenic diagnosis of sepsis, while their sensitivities have always been unsatisfactory for the usually low concentration of pathogens in the blood of sepsis patients. In this study, we established a fast diagnostic method using magnetic beads coated with human recombined mannose-binding lectin that makes it possible to concentrate pathogens from human plasma that have low concentrations of pathogens. With subsequent microculture (MC) and real-time PCR, this method allowed the detection of 1-10 CFUs/ml of Staphylococcus aureus, Group A Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Candida tropicalis, or C. albicans from human plasma within 9.5 h, which was 21-80 h earlier than blood culture. The combination of pathogen enrichment and MC made the detection of sepsis-causing pathogens more time-saving and more sensitive than blood culture or real-time PCR alone.

A rapid multiplex real-time PCR detection of toxigenic Clostridioides difficile directly from fecal samples.

This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/µL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03434-6.

Preliminary Study of the Protectiveness of Vaccination Against the COVID-19 in the Outbreak of VOC Omicron BA.2 - Jilin City, Jilin Province, China, March 3-April 12, 2022.

What is already known about this topic?: 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. What is added by this report?: 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. What are the implications for public health practice?: 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.

Super Dominant Pathobiontic Bacteria in the Nasopharyngeal Microbiota Cause Secondary Bacterial Infection in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease responsible for many infections worldwide. Differences in respiratory microbiota may correlate with disease severity. Samples were collected from 20 severe and 51 mild COVID-19 patients. High-throughput sequencing of the 16S rRNA gene was used to analyze the bacterial community composition of the upper and lower respiratory tracts. The indices of diversity were analyzed. When one genus accounted for >50% of reads from a sample, it was defined as a super dominant pathobiontic bacterial genus (SDPG). In the upper respiratory tract, uniformity indices were significantly higher in the mild group than in the severe group (P < 0.001). In the lower respiratory tract, uniformity indices, richness indices, and the abundance-based coverage estimator were significantly higher in the mild group than in the severe group (P < 0.001). In patients with severe COVID-19, SDPGs were detected in 40.7% of upper and 63.2% of lower respiratory tract samples. In patients with mild COVID-19, only 10.8% of upper and 8.5% of lower respiratory tract samples yielded SDPGs. SDPGs were present in both upper and lower tracts in seven patients (35.0%), among which six (30.0%) patients possessed the same SDPG in the upper and lower tracts. However, no patients with mild infections had an SDPG in both tracts. Staphylococcus, Corynebacterium, and Acinetobacter were the main SDPGs. The number of SDPGs identified differed significantly between patients with mild and severe COVID-19 (P < 0.001). SDPGs in nasopharyngeal microbiota cause secondary bacterial infection in COVID-19 patients and aggravate pneumonia. IMPORTANCE The nasopharyngeal microbiota is composed of a variety of not only the true commensal bacterial species but also the two-face pathobionts, which are one a harmless commensal bacterial species and the other a highly invasive and deadly pathogen. In a previous study, we found that the diversity of nasopharyngeal microbiota was lost in severe influenza patients. We named the genus that accounted for over 50% of microbiota abundance as super dominant pathobiontic genus, which could invade to cause severe pneumonia, leading to high fatality. Similar phenomena were found here for SARS-CoV-2 infection. The diversity of nasopharyngeal microbiota was lost in severe COVID-19 infection patients. SDPGs in nasopharyngeal microbiota were frequently detected in severe COVID-19 patients. Therefore, the SDPGs in nasopharynx microbiota might invade into low respiratory and be responsible for secondary bacterial pneumonia in patients with SARS-CoV-2 infection.

A narrative review of Clostridioides difficile infection in China.

Clostridioides difficile is the predominant pathogen responsible for antimicrobial associated diarrhea (AAD) and health care facility-associated infectious diarrhea. The role of C. difficile in China and its impact on public health have gained attention in recent years. Most clinical C. difficile isolates in China belong to multilocus sequence type clade 1 with sequence types (STs) 3, 35 and 54 predominating. Of note, the proportion of C. difficile isolates from clade 4, especially ST37 (PCR ribotype 17), is much higher in China than in other areas. In China, the antimicrobial-resistance profile of C. difficile is similar to that of other countries, demonstrating a higher resistance rate to erythromycin, clindamycin, and fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin). In general, susceptibility to vancomycin and metronidazole of clinical C. difficile in China is high, however, some resistance to metronidazole have recently been reported. Preclinical research on C. difficile in animals in China is limited, and different studies have reported varied isolation rates and antimicrobial resistance profiles. The diverse molecular types of C. difficile in China merit further epidemiological, genomic and evolutionary investigation. While the use of probiotics in preventing C. difficile infection (CDI) have received both support and opposition, the discovery of new probiotics and new formulations are showing promising results in combating the threat posed by CDI.

A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology.

The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 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. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), 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 cycle threshold (CT) values less than 27 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. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.

A COVID-19 Outbreak - Nangong City, Hebei Province, China, January 2021.

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.

Expression of Serum miR-155 in Children with Mycoplasma pneumoniae Pneumonia and Its Role in Immunity to Mycoplasma pneumoniae.

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.

New ribotype Clostridioides difficile from ST11 group revealed higher pathogenic ability than RT078.

Clostridioides difficile is the predominant antibiotic-associated enteropathogen associated with diarrhoea or pseudomembranous colitis in patients worldwide. Previously, we identified C. difficile RT078 isolates (CD21062) from elderly patients in China, including two new ribotype strains (CD10010 and CD12038) belonging to the ST11 group, and their genomic features were also investigated. This study compared sporulation, spore germination, toxin expression, flagellar characteristics, and adhesion among these strains in vitro and analysed their pathogenic ability in vivo using animal models. The results showed sporulation and spore germination did not significantly differ among the three C. difficile strains. CD10010 and CD12038 showed higher transcriptional levels of toxins until 48 h; thereafter, the transcriptional levels of toxins remained constant among RT078, CD10010, and CD12038. RT078 showed a loss of flagellum and its related genes, whereas CD12038 showed the highest motility in vitro. Both CD10010 and CD12038 initially showed flg phase OFF, and the flagellar switch reversed to phase ON after 48 h in swim agar. Flagellar proteins and toxins were both upregulated when flg phase OFF changed to flg phase ON status, enhancing their pathogenic ability. CD12038 showed the highest adhesion to Hep-2 cells. Histopathology and inflammation scores demonstrated that CD12038 caused the most severe tissue damage and infection in vivo. The new ribotype strains, particularly CD12038, exhibit higher pathogenic ability than the typical RT078 strain, both in vitro and in vivo. Therefore, more attention should be paid to this new C. difficile strain in epidemiological research; further studies are warranted.

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.

Response of the gut microbiota during the Clostridioides difficile infection in tree shrews mimics those in humans.

BACKGROUND: Clostridioides difficile is a major cause of antibiotic associated diarrhea. Several animal models are used to study C. difficile infection (CDI). The tree shrew has recently been developed as a model of primate processes. C. difficile infection has not been examined in tree shrews. We infected tree shrews with hyper-virulent C. difficile strains and examined the alterations in gut microbiota using 16S rRNA gene sequencing. RESULTS: C. difficile colonized the gastrointestinal tract of tree shrew and caused diarrhea and weight loss. Histopathologic examination indicated structures and mucosal cell destruction in ileal and colonic tissues. The gut microbial community was highly diversity before infection and was dominated by Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Antibiotic administration decreased the diversity of the gut microbiota and led to an outgrowth of Lactobacillus. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia, Blautia, and Tyzzerella increased following C. difficile infection. These taxa could be biomarkers for C. difficile colonization. CONCLUSIONS: In general, the disease symptoms, histopathology, and gut microbiota changes following C. difficile infection in tree shrews were similar to those observed in humans.

Development of an inactivated vaccine candidate for SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.

The molecular characters and antibiotic resistance of Clostridioides difficile from economic animals in China.

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.

Direct Blood Culturing of Candida spp. on Solid Medium by a Rapid Enrichment Method with Magnetic Beads Coated with Recombinant Human Mannan-Binding Lectin.

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 <1 CFU/ml (an average of 29 h earlier). Additionally, the clinical sensitivity of the M1 method was 90.5% compared with that of the standard blood culture method when detecting frozen plasma from patients. More significantly, the turnaround time of the M1 method for blood culture could be reduced by approximately 37 to 43 h compared with that of the standard blood culture method in clinical sample identification.

Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing.

BACKGROUND: Clade 5 Clostridioides difficile diverges 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 ST11 C. difficile isolates, 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. RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, 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. CONCLUSIONS: We 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.

Antibiotic resistance of clinical isolates of Clostridioides difficile in China and its association with geographical regions and patient age.

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.

Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay.

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.

Independent Microevolution Mediated by Mobile Genetic Elements of Individual Clostridium difficile Isolates from Clade 4 Revealed by Whole-Genome Sequencing.

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.