Simultaneous Detection of Helicobacter pylori and Clarithromycin Resistance Mutations Using RAA-CRISPR/Cas13a Assay.

Background: Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance. Methods: We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency. Results: We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/µL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing. Conclusion: SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.

Detection of Staphylococcus aureus virulence gene pvl based on CRISPR strip.

Introduction: Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for both hospital-acquired and community-acquired infections. Among its arsenal of virulence factors, Panton-Valentine Leucocidin (PVL) is closely associated with severe diseases such as profound skin infections and necrotizing pneumonia. Patients infected with pvl-positive S. aureus often exhibit more severe symptoms and carry a substantially higher mortality risk. Therefore, it is crucial to promptly and accurately detect pvl-positive S. aureus before initiating protective measures and providing effective antibacterial treatment. Methods: In this study, we propose a precise identification and highly sensitive detection method for pvl-positive S. aureus based on recombinase-assisted amplification and the CRISPR-ERASE strip which we previously developed. Results: The results revealed that this method achieved a detection limit of 1 copy/µL for pvl-positive plasmids within 1 hour. The method successfully identified all 25 pvl-positive and 51 pvl-negative strains among the tested 76 isolated S. aureus samples, demonstrating its concordance with qPCR. Discussion: These results show that the CRISPR-ERASE detection method for pvl-positive S. aureus has the advantages of high sensitivity and specificity, this method combines the characteristics of recombinase-assisted amplification at room temperature and the advantages of ERASE test strip visualization, which can greatly reduce the dependence on professional laboratories. It is more suitable for on-site detection than PCR and qPCR, thereby providing important value for rapid on-site detection of pvl.

A highly susceptible hACE2-transgenic mouse model for SARS-CoV-2 research.

Several animal models have been used to assist the development of vaccines and therapeutics since the COVID-19 outbreak. Due to the lack of binding affinity of mouse angiotensin-converting enzyme II (ACE2) to the S protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), increasing the susceptibility of mice to SARS-CoV-2 infection was considered in several ways. Here, we generated a COVID-19 mouse model expressing human ACE2 (hACE2) under the control of the CAG promoter. Overexpression of hACE2 did not pose a significant effect on weight growth. After SARS-CoV-2 inoculation, mice showed obvious viral replication and production of inflammation within 7 days, with a gradual decrease in body weight until death. Virological testing found that the virus can replicate in the respiratory system, small intestine, and brain. Additionally, this mouse model was applied to compare two antibody drug candidates, the anti-RBD antibody (MW06) and the mouse CD24-conjugated anti-RBD antibody (mCD24-MW06). Differences in antiviral effects between these two antibodies can be demonstrated in this mouse model when a challenge dose that invalidates the anti-RBD antibody treatment was used. This study provided a new mouse model for studying SARS-CoV-2 pathogenesis and evaluating potential interventions.

Short-Term Exposure to Ambient Air Pollution and Influenza: A Multicity Study in China.

BACKGROUND: Air pollution is a major risk factor for planetary health and has long been suspected of predisposing humans to respiratory diseases induced by pathogens like influenza viruses. However, epidemiological evidence remains elusive due to lack of longitudinal data from large cohorts. OBJECTIVE: Our aim is to quantify the short-term association of influenza incidence with exposure to ambient air pollutants in Chinese cities. METHODS: Based on air pollutant data and influenza surveillance data from 82 cities in China over a period of 5 years, we applied a two-stage time series analysis to assess the association of daily incidence of reported influenza cases with six common air pollutants [particulate matter with aerodynamic diameter ≤2.5µm (PM2.5), particulate matter with aerodynamic diameter ≤10µm (PM10), NO2, SO2, CO, and O3], while adjusting for potential confounders including temperature, relative humidity, seasonality, and holiday effects. We built a distributed lag Poisson model for one or multiple pollutants in each individual city in the first stage and conducted a meta-analysis to pool city-specific estimates in the second stage. RESULTS: A total of 3,735,934 influenza cases were reported in 82 cities from 2015 to 2019, accounting for 72.71% of the overall case number reported in the mainland of China. The time series models for each pollutant alone showed that the daily incidence of reported influenza cases was positively associated with almost all air pollutants except for ozone. The most prominent short-term associations were found for SO2 and NO2 with cumulative risk ratios of 1.094 [95% confidence interval (CI): 1.054, 1.136] and 1.093 (95% CI: 1.067, 1.119), respectively, for each 10 µg/m3 increase in the concentration at each of the lags of 1-7 d. Only NO2 showed a significant association with the daily incidence of influenza cases in the multipollutant model that adjusts all six air pollutants together. The impact of air pollutants on influenza was generally found to be greater in children, in subtropical cities, and during cold months. DISCUSSION: Increased exposure to ambient air pollutants, particularly NO2, is associated with a higher risk of influenza-associated illness. Policies on reducing air pollution levels may help alleviate the disease burden due to influenza infection. https://doi.org/10.1289/EHP12146.

Highly sensitive and rapid point-of-care testing for HIV-1 infection based on CRISPR-Cas13a system.

BACKGROUND: Human immunodeficiency virus type one (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS). AIDS remains a global public health concern but can be effectively suppressed by life-long administration of combination antiretroviral therapy. Early detection and diagnosis are two key strategies for the prevention and control of HIV/AIDS. Rapid and accurate point-of-care testing (POCT) provides critical tools for managing HIV-1 epidemic in high-risk areas and populations. METHODS: In this study, a POCT for HIV-1 RNA was developed by CRISPR-Cas13a lateral flow strip combined with reverse transcriptase recombinase-aided amplification (RT-RAA) technology, the results can be directly observed by naked eyes. RESULTS: Moreover, with the degenerate base-binding CRISPR-Cas13a system was introduced into the RT-RAA primer designing, the technology developed in this study can be used to test majority of HIV-1 RNA with limit of detection (LOD) 1 copy/µL, while no obvious cross-reaction with other pathogens. We evaluated this method for detecting HIV-1 RNA of clinical samples, the results showed that the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were 91.81% (85.03- 96.19%), 100% (92.60-100%), 100% (96.41-100%), 39.14% (25.59-54.60%) and 92.22% (86.89-95.88%), respectively. The lowest viral load detectable by this method was 112copies/mL. CONCLUSION: Above all, this method provides a point-of-care detection of HIV-1 RNA, which is stable, simple and with good sensitivity and specificity. This method has potential to be developed for promoting early diagnosis and treatment effect monitoring of HIV patients in clinical.

Imunocapture Magnetic Beads Enhanced and Ultrasensitive CRISPR-Cas13a-Assisted Electrochemical Biosensor for Rapid Detection of SARS-CoV-2.

The rapid and ongoing spread of the coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emphasizes the urgent need for an easy and sensitive virus detection method. Here, we describe an immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, collectively known as CRISPR-Cas13a technology. At the core of the detection process, low-cast and immobilization-free commercial screen-printed carbon electrodes are used to measure the electrochemical signal, while streptavidin-coated immunocapture magnetic beads are used to reduce the background noise signal and enhance detection ability by separating the excessive report RNA, and a combination of isothermal amplification methods in the CRISPR-Cas13a system is used for nucleic acid detection. The results showed that the sensitivity of the biosensor increased by two orders of magnitude when the magnetic beads were used. The proposed biosensor required approximately 1 h of overall processing time and demonstrated an ultrasensitive ability to detect SARS-CoV-2, which could be as low as 1.66 aM. Furthermore, owing to the programmability of the CRISPR-Cas13a system, the biosensor can be flexibly applied to other viruses, providing a new approach for powerful clinical diagnostics.

A rapid and efficient platform for antiviral crRNA screening using CRISPR-Cas13a-based nucleic acid detection.

Introduction: Rapid and high-throughput screening of antiviral clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) is urgently required for the CRISPR-Cas13a antiviral system. Based on the same principle, we established an efficient screening platform for antiviral crRNA through CRISPR-Cas13a nucleic acid detection. Method: In this study, crRNAs targeting PA, PB1, NP, and PB2 of the influenza A virus (H1N1) were screened using CRISPR-Cas13a nucleic acid detection, and their antiviral effects were confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RNA secondary structures were predicted by bioinformatics methods. Results: The results showed that crRNAs screened by CRISPR-Cas13a nucleic acid detection could effectively inhibit viral RNA in mammalian cells. Besides, we found that this platform for antiviral crRNA screening was more accurate than RNA secondary structure prediction. In addition, we validated the feasibility of the platform by screening crRNAs targeting NS of the influenza A virus (H1N1). Discussion: This study provides a new approach for screening antiviral crRNAs and contributes to the rapid advancement of the CRISPR-Cas13a antiviral system.

CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation.

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas) gene editing can induce P53 activation, large genome fragment deletions, and chromosomal structural variations. Here, gene expression was detected in host cells using transcriptome sequencing following CRISPR/Cas9 gene editing. We found that the gene editing reshaped the gene expression, and the number of differentially expressed genes was correlated with the gene editing efficiency. Moreover, we found that alternative splicing occurred at random sites and that targeting a single site for gene editing may not result in the formation of fusion genes. Further, gene ontology and KEGG enrichment analysis showed that gene editing altered the fundamental biological processes and pathways associated with diseases. Finally, we found that cell growth was not affected; however, the DNA damage response protein-γH2AX-was activated. This study revealed that CRISPR/Cas9 gene editing may induce cancer-related changes and provided basic data for research on the safety risks associated with the use of the CRISPR/Cas9 system.

A randomized, double-blind, placebo-controlled phase 1 and phase 2 clinical trial to evaluate efficacy and safety of a SARS-CoV-2 vaccine SCoK in adults.

BACKGROUND: To determine an appropriate dose of, and immunization schedule for, a vaccine SCoK against COVID-19 for an efficacy study; herein, we conducted randomized controlled trials to assess the immunogenicity and safety of this vaccine in adults. METHODS: These randomized, double-blind, placebo-controlled phase 1 and 2 trials of vaccine SCoK were conducted in Binhai District, Yan City, Jiangsu Province, China. Younger and older adult participants in phase 1 and 2 trials were sequentially recruited into different groups to be intramuscularly administered 20 or 40 µg vaccine SCoK or placebo. Participants were enrolled into our phase 1 and 2 studies to receive vaccine or placebo. RESULTS: No serious vaccine-related adverse events were observed in either trial. In both trials, local and systemic adverse reactions were absent or mild in most participants. In our phase 1 and 2 studies, the vaccine induced significantly increased neutralizing antibody responses to pseudovirus and live SARS-CoV-2. The vaccine induced significant neutralizing antibody responses to live SARS-CoV-2 on day 14 after the last immunization, with NT50s of 80.45 and 92.46 in participants receiving 20 and 40 µg doses, respectively; the seroconversion rates were 95.83% and 100%. The vaccine SCoK showed a similar safety and immunogenicity profiles in both younger participants and older participants. The vaccine showed better immunogenicity in phase 2 than in phase 1 clinical trial. Additionally, the incidence of adverse reactions decreased significantly in phase 2 clinical trial. The vaccine SCoK was well tolerated and immunogenic.

Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis.

Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.

Coxiella burnetii Plasmid Effector B Promotes LC3-II Accumulation and Contributes To Bacterial Virulence in a SCID Mouse Model.

Coxiella burnetii, the causative agent of zoonotic Q fever, is characterized by replicating inside the lysosome-derived Coxiella-containing vacuole (CCV) in host cells. Some effector proteins secreted by C. burnetii have been reported to be involved in the manipulation of autophagy to facilitate the development of CCVs and bacterial replication. Here, we found that the Coxiella plasmid effector B (CpeB) localizes on vacuole membrane targeted by LC3 and LAMP1 and promotes LC3-II accumulation. Meanwhile, the C. burnetii strain lacking the QpH1 plasmid induced less LC3-II accumulation, which was accompanied by smaller CCVs and lower bacterial loads in THP-1 cells. Expression of CpeB in the strain lacking QpH1 led to restoration in LC3-II accumulation but had no effect on the smaller CCV phenotype. In the severe combined immune deficiency (SCID) mouse model, infections with the strain expressing CpeB led to significantly higher bacterial burdens in the spleen and liver than its parent strain devoid of QpH1. We also found that CpeB targets Rab11a to promote LC3-II accumulation. Intratracheally inoculated C. burnetii resulted in lower bacterial burdens and milder lung lesions in Rab11a conditional knockout (Rab11a-/- CKO) mice. Collectively, these results suggest that CpeB promotes C. burnetii virulence by inducing LC3-II accumulation via a pathway involving Rab11a.

Highly Sensitive Detection Method for HV69-70del in SARS-CoV-2 Alpha and Omicron Variants Based on CRISPR/Cas13a.

As SARS-CoV-2 variants continue to evolve, identifying variants with adaptive diagnostic tool is critical to containing the ongoing COVID-19 pandemic. Herein, we establish a highly sensitive and portable on-site detection method for the HV69-70del which exist in SARS-CoV-2 Alpha and Omicron variants using a PCR-based CRISPR/Cas13a detection system (PCR-CRISPR). The specific crRNA (CRISPR RNA) targeting the HV69-70del is screened using the fluorescence-based CRISPR assay, and the sensitivity and specificity of this method are evaluated using diluted nucleic acids of SARS-CoV-2 variants and other pathogens. The results show that the PCR-CRISPR detection method can detect 1 copies/µL SARS-CoV-2 HV69-70del mutant RNA and identify 0.1% of mutant RNA in mixed samples, which is more sensitive than the RT-qPCR based commercial SARS-CoV-2 variants detection kits and sanger sequencing. And it has no cross reactivity with ten other pathogens nucleic acids. Additionally, by combined with our previously developed ERASE (Easy-Readout and Sensitive Enhanced) lateral flow strip suitable for CRISPR detection, we provide a novel diagnosis tool to identify SARS-CoV-2 variants in primary and resource-limited medical institutions without professional and expensive fluorescent detector.

Dissemination of blaNDM-5 in Escherichia coli through the IncX3 Plasmid from Different Regions in China.

The spread of NDM-5-producing Escherichia coli has become a severe challenge in clinical therapy, which necessitates reliable detection and surveillance methods. However, limited information is available regarding the prevalence and dissemination of the blaNDM-5 gene in E. coli in China. Therefore, we investigated the dissemination of the blaNDM-5 gene in carbapenem-resistant E. coli isolates from different regions. A total of 1,180 carbapenem-resistant enterobacteriaceae strains were obtained from patients admitted to the 20 sentinel hospitals in 8 cities. Strains positive for blaNDM-5 were detected using the Vitek 2 compact system, 16S ribosomal RNA (rRNA) gene sequencing, polymerase chain reaction, the S1 pulsed-field gel electrophoresis assay, and Southern blot hybridization. The horizontal-transfer capability of the blaNDM gene was assessed by filter mating with a standard E. coli J53 azide-resistant strain as the recipient. Genotyping, susceptibility testing, and whole genome sequencing were performed. Seven strains of blaNDM-5-positive E. coli were detected in 1,180 clinical strains from different regions in China. The blaNDM-5-carrying strains showed resistance to multiple tested antibiotics and belonged to two widespread sequence types, sequence type (ST)167 and ST405. Antimicrobial resistance genes, including blaCTX-M, blaOXA, blaCMY, and two novel blaTEM variants (blaTEM-230 and blaTEM-231) were also identified. Southern blotting located the blaNDM-5 gene on 46 kb IncX3 plasmids in all isolates, which showed only two single nucleotide differences between EJN003 and the other strains. This study further confirms the increasing occurrence of blaNDM-5-carrying IncX3 plasmids and the dissemination of carbapenem resistance in E. coli isolates using the plasmid from different parts in China, which warrants stringent surveillance and control measures.

An experimental method for efficiently evaluating the size-resolved sampling efficiency of liquid-absorption aerosol samplers.

Aerosol samplers are critical tools for studying indoor and outdoor aerosols. Development and evaluation of samplers is often labor-intensive and time-consuming due to the need to use monodisperse aerosols spanning a range of sizes. This study develops a rapid experimental methodology using polydisperse solid aerosols to evaluate size-resolved aerosol-to-aerosol (AtoA) and aerosol-to-hydrosol (AtoH) sampling efficiencies. Arizona Test Dust (diameter 0.5-20 µm) was generated and dispersed into an aerosol test chamber and two candidate samplers were tested. For the AtoA test, aerosols upstream and downstream of a sampler were measured using an online aerodynamic particle sizer. For the AtoH test, aerosols collected in sampling medium were mixed with a reference sample and then measured by the laser diffraction method. The experimental methodology were validated as an impressive time-saving procedure, with reasonable spatial uniformity and time stability of aerosols in the test chamber and an acceptable accuracy of absolute mass quantification of collected particles. Evaluation results showed that the AGI-30 and the BioSampler sampler had similar size-resolved sampling efficiencies and that efficiencies decreased with decreasing sampling flow rate. The combined evaluation of AtoA and AtoH efficiency provided more comprehensive performance indicators than either test alone. The experimental methodology presented here can facilitate the design and choice of aerosol sampler.

DANMEL: A manually curated reference database for analyzing mobile genetic elements associated with bacterial drug resistance.

We have developed a manually curated online reference database, DANMEL (http://124.239.252.254/danmel/), that addresses the lack of accurate dissection and annotation of the genetic structures of mobile genetic elements (MGEs) with genes for drug resistance. DANMEL contains accurately annotated and genetically dissected reference MGEs covering 5 categories and 135 subcategories/subfamilies of MGEs. Further, DANMEL provides a detailed guide on how to precisely annotate MGEs. DANMEL also provides SEARCH/BLAST functions to facilitate finding reference MGEs. Overall, DANMEL will aid researchers to conduct in-depth genetic analysis of sequenced bacterial MGEs with drug-resistance genes and further facilitate a better understanding of bacterial MGEs associated with drug resistance at a genomic level.

The Establishment and Application of Mobile Electronic Surveillance System for Infectious Diseases with the Help of China - Sierra Leone, 2016-Present.

INTRODUCTION: Infectious disease surveillance has long been a challenge for low-income countries like Sierra Leone. Traditional approaches based on paper and Short Message Service (SMS) were subject to severe delays in obtaining, transmitting, and analyzing information. METHODS: During the China aid operation for fighting Ebola since the end of 2014, a mobile electronic surveillance system for infectious diseases (MESSID) was developed in collaboration with the Republic of Sierra Leone Armed Forces (RSLAF), which comprised an Android-based reporting system and a complementary web-based program designed by Active Server Page.NET (ASP.NET) with the main functions including surveillance, real-time reporting, and risk assessment of infectious diseases. RESULTS: MESSID was successfully registered in June 2016 and had been used by all medical and health institutions in RSLAF. From June 1, 2016 to July 5, 2021, 34,419 cases were diagnosed with 47 infectious diseases of 5 categories, with a total of 42 clinical symptoms. Compared to traditional approaches based on paper and SMS, the MESSID showed flexibility, high efficiency, convenience, and acceptability. DISCUSSION: MESSID is an accessible tool for surveillance of infectious diseases in Sierra Leone and possibly in other African countries with similar needs, capable of improving timeliness of disease reporting, thus rendering a timely outbreak detection and response.

Sensitive and Easy-Read CRISPR Strip for COVID-19 Rapid Point-of-Care Testing.

Rapid and clinically sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) play an important role in the contact tracing and containment of the COVID-19 pandemic. A recently developed field-deployable clustered regularly interspaced short palindromic repeats (CRISPR) detection assay with lateral flow strips shows promise for point-of-care detection of SARS-CoV-2. However, the limit of detection of paper strip-based assays (10-100 copies/µL) is much lower than that of fluorescence-based detection methods. In this study, we developed an easy-readout and sensitive enhanced (ERASE) strip to visualize the results of CRISPR detection and improve the sensitivity to 1 copy/µL with an unambiguous easy-read result. Using 649 clinical samples from blind specimens collected from patients in China, we validated our ERASE assay for SARS-CoV-2 RNA detection with 90.67% positive predictive agreement and 99.21% negative predictive agreement. In conclusion, our study provided a customized CRISPR strip for use in a simple, rapid, ultrasensitive, and highly specific assay for SARS-CoV-2 detection. (Clinical Trial Registration number: 2020-008-01; [2020]IEC(ZD01); PJ-NBEY-2020-009-01; 2020#34).

A new plasmid carrying mphA causes prevalence of azithromycin resistance in enterotoxigenic Escherichia coli serogroup O6.

BACKGROUND: At present, azithromycin has become an effective treatment for severe diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) infection. However, enterobacteria have begun to develop resistance to azithromycin and have attracted attention in recent years. This study conducted to described the emergence of a high proportion of azithromycin-resistant ETEC serogroup O6 strains in Shanghai and to analyzed the mechanisms of azithromycin resistance. RESULTS: Strains from adult diarrhea patients with ETEC serogroup O6 infections were collected by Shanghai Diarrhea Surveillance Network and the Foodborne Surveillance Network from 2016 to 2018. We tested 30 isolates of ETEC O6 serogroup, 26 of which were resistant to azithromycin. Phylogenetic analysis revealed that these ETEC serogroup O6 strains have formed an independent dominant clone. S1-PFGE and southern blotting revealed the presence of the mphA gene on the 103 kb plasmid. Illumina and Nanopore sequencing and plasmid coverage analysis further confirmed that azithromycin-resistant strains carried a novel IncFII plasmid harboring mphA and blaTEM-1 resistance genes. CONCLUSIONS: This is the first study to report a high proportion of azithromycin resistance in a particular ETEC serogroup due to a specific plasmid carrying mphA. Our findings indicate the rapid spread of azithromycin resistance, highlighting the urgency of stringent surveillance and control measure.

The phenotypic and molecular characteristics of antimicrobial resistance of Salmonella enterica subsp. enterica serovar Typhimurium in Henan Province, China.

BACKGROUND: Salmonella enterica subsp. enterica serovar Typhimurium infections continue to be a significant public health threat worldwide. The aim of this study was to investigate antibiotic resistance among 147 S. Typhimurium isolates collected from patients in Henan, China from 2006 to 2015. METHODS: 147 S. Typhimurium isolates were collected from March 2006 to November 2015 in Henan Province, China. Antimicrobial susceptibility testing was performed, and the resistant genes of ciprofloxacin, cephalosporins (ceftriaxone and cefoxitin) and azithromycin were detected and sequenced. Clonal relationships were assessed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: Of the 147 isolates, 91.1% were multidrug resistant (MDR), with 4.1% being resistant to all antibiotic classes tested. Of concern, 13 MDR isolates were co-resistant to the first-line treatments cephalosporins and ciprofloxacin, while three were also resistant to azithromycin. Seven PFGE patterns were identified among the 13 isolates. All of the isolates could be assigned to one of four main groups, with a similarity value of 89%. MLST assigned the 147 isolates into five STs, including two dominant STs (ST19 and ST34). Of the 43 ciprofloxacin-resistant isolates, 39 carried double gyrA mutations (Ser83Phe, Asp87Asn/Tyr/Gly) and a single parC (Ser80Arg) mutation, including 1 isolate with four mutations (gyrA: Ser83Phe, Asp87Gly; parC: Ser80Arg; parE: Ser458Pro). In addition, 12 isolates not only carried mutations in gyrA and parC but also had at least one plasmid-mediated quinolone resistance (PMQR) gene. Among the 32 cephalosporin-resistant isolates, the most common extended-spectrum ß-lactamase (ESBL) gene was blaOXA-1, followed by blaCTX-M, blaTEM-1, and blaCMY-2. Moreover, the mphA gene was identified in 5 of the 15 azithromycin-resistant isolates. Four MDR isolates contained ESBL and PMQR genes, and one of them also carried mphA in addition. CONCLUSION: The high level of antibiotic resistance observed in S. Typhimurium poses a great danger to public health, so continuous surveillance of changes in antibiotic resistance is necessary.