OBJECTIVE To investigate the distribution of drug-resistant bacteria on surfaces of highly frequent contact objects in ICUs so as to provide scientific basis for prevention and control of nosocomial infection and provide guidance for disinfection in hospitals. METHODS The surfaces of highly frequent contact objects were sampled from respiratory medicine department from Jul 2021 to Dec 2021. The drug-resistant strains were screened and cultured by 5 types of antibiotic media, and the species of the strains were identified by means of PCR amplification of full-length 16S rRNA gene and first generation sequencing technology. RESULTS A total of 48 environmental swabs of 6 batches were collected from 8 highly frequent contact sites in the ICUs, 35 of which were cultured positive, with the total positive rate 72.92%. Totally 115 strains of bacteria were screened, isolated and identified. The top 3 species of bacteria were as follows: Acinetobacter baumannii（14 strains, 12.17%）, Pseudomonas aeruginosa（13 strains, 11.30%）, Klebsiella pneumoniae（11 strains, 9.57%）. The sampled sites with heavy loads of drug-resistant bacteria were as the following: sink traps（30 strains）, faucets（25 strains）, bedside tables（20 strains） and bed railings（18 strains）; 4（13.33%） strains of P. aeruginosa and 4（13.33%） strains of K. pneumoniae were isolated from sink traps; 4（16%） strains of Acinetobacter berghei and 4（16%） strains of P. aeruginosa were isolated from faucets; 2（10%） strains of P. aeruginosa were isolated from bedside tables; totally 18 strains of drug-resistant bacteria were isolated from bed railings, A. baumannii（6 strains, 33.33%） was dominant. CONCLUSION There is difference in the distribution of drug-resistant bacteria isolated from the surfaces of objects in the ICUs. There are more drug-resistant strains on the sampled sites close to water sources and patients. It is suggested that the hospital should strengthen the disinfection of surfaces of objects in ICUs so as to effectively control the nosocomial drug-resistant bacteria infections.

Introduction Influenza A virus (IAV)-induced dysbiosis may predispose to severe bacterial superinfections. Most studies have focused on the microbiota of single mucosal surfaces; consequently, the relationships between microbiota at different anatomic sites in IAV-infected mice have not been fully studied. Methods We characterized respiratory and gut microbiota using full-length 16S rRNA gene sequencing by Nanopore sequencers and compared the nasopharyngeal, oropharyngeal, lung and gut microbiomes in healthy and IAV-infected mice. Results The oropharyngeal, lung and gut microbiota of healthy mice were dominated by Lactobacillus spp., while nasopharyngeal microbiota were comprised primarily of Streptococcus spp. However, the oropharyngeal, nasopharyngeal, lung, and gut microbiota of IAV-infected mice were dominated by Pseudomonas, Escherichia , Streptococcus , and Muribaculum spp., respectively. Lactobacillus murinus was identified as a biomarker and was reduced at all sites in IAV-infected mice. The microbiota composition of lung was more similar to that of the nasopharynx than the oropharynx in healthy mice. Discussion These findings suggest that the main source of lung microbiota in mice differs from that of adults. Moreover, the similarity between the nasopharyngeal and lung microbiota was increased in IAV-infected mice. We found that IAV infection reduced the similarity between the gut and oropharyngeal microbiota. L. murinus was identified as a biomarker of IAV infection and may be an important target for intervention in post-influenza bacterial superinfections.

The respiratory microbiome plays an important role in respiratory tract infections. In this study, we characterized the URT and LRT microbiota, the host immune response, and plasma metabolic profiles during IAV-MRSA coinfection and evaluated their correlations.

Klebsiella pneumoniae often causes life-threatening infections in patients globally. Despite its notability, little is known about potential nosocomial outbreak and spread of K. pneumoniae among pediatric patients in low- and middle-income countries. Ninety-eight K. pneumoniae strains isolated from pediatric patients in a large general hospital in China between February 2018 and May 2019 were subjected to nanopore and Illumina sequencing and genomic analysis to elucidate transmission and genetic diversity. The temporal distribution patterns of K. pneumoniae revealed a cluster of sequence type 11 (ST11) strains comprising two clades. Most inferred transmissions were of clade 1, which could be traced to a common ancestor dating to mid-2017. An infant in the coronary care unit played a central role, potentially seeding transmission clusters in other wards. Major genomic changes during the outbreak included chromosomal mutations associated with virulence and gains and losses of plasmids encoding resistance. In summary, we report a nosocomial outbreak among pediatric patients caused by clonal dissemination of KPC-2-producing ST11 K. pneumoniae. Our findings highlight the value of whole-genome sequencing during outbreak investigations and illustrate that transmission chains can be identified during hospital stays. IMPORTANCE We report a nosocomial outbreak among pediatric patients caused by clonal dissemination of blaKPC-2-carrying ST11 K. pneumoniae. Strains of various sequence types coexist in the complex hospital environment; the quick emergence and spread of ST11 strains were mainly due to the plasmid-mediated acquisition of resistance genes. The spread of hospital infection was highly associated with several specific wards, suggesting the importance of genomic surveillance on wards at high risk of infection.

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a bla(NDM-1) gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two bla(NDM-1) gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-bla(NDM-1)-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without bla(NDM-1) copy number changes. This study characterizes two novel bla(NDM-1)-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

Objective: Fever with thrombocytopenia syndrome virus (SFTS) is a tick-borne infection now known to spread among humans as an aerosol, which has resulted in several outbreaks across Asia over the past decade. As mortality is substantial, it is vital to establish a rapid, on-site nucleic acid detection method for diagnosis. Here we describe such a method for SFTSV (Dabie bandavirus) based on CRISPR-Cas13a. Methods: Specific recombinase-aided amplification (RAA) primers and CRISPR (cr)RNA nucleic acid detection targets were designed and synthesized for the conserved sequence of the SFTSV genome, and fluorescent CRISPR detection was used to screen for high-sensitivity crRNAs. Colloidal immunochromatography test paper was used to read CRISPR detection results. Sensitivity and specificity were evaluated by running tests on gradient dilutions of SFTSV nucleic acid and the nucleic acids of other pathogens with similar transmission routes or clinical manifestations. Results: One crRNA with high detection sensitivity was screened out of 5 crRNAs with conserved sequences from the SFTSV genome. This CRISPR nucleic acid-based detection method was able to detect a single crRNA copy per microliter but not the nucleic acids of similar pathogens. Conclusion: This CRISPR test strip detection method permits rapid, sensitive, and specific diagnosis of SFTS without the need for advanced nucleic acid detection equipment, thus allowing for on-site application.

Pseudomonas aeruginosa, a common opportunistic pathogen, is one of the leading etiological agents of nosocomial infections. Many previous studies have reported the nosocomial transmission and epidemiology of P. aeruginosa infections. However, longitudinal studies regarding the dynamics of P. aeruginosa colonization and infection in health care settings are limited. We obtained longitudinal samples from aged patients with prolonged intensive care unit (ICU) stays (similar to 4 to 19 months). P. aeruginosa was isolated from 71 samples obtained from seven patients and characterized by whole-genome sequencing. The P. aeruginosa isolates were assigned to 10 clonal complexes, and turnover of main clones was observed in sequential sputum samples from two patients. By comparing intraclonal genomic diversities, we identified two clones that had significantly higher numbers of single nucleotide polymorphisms and variations in homopolymeric sequences than the other clones, indicating a hypermutator phenotype. These hypermutator clones were associated with mutations T147I/G521S and P27L in the MutL protein, and their mutation rates were estimated to be 3.20 x 10(-5) and 6.59 x 10(-5) per year per nucleotide, respectively. We also identified 24 recurrently mutated genes that exhibited intraclonal diversity in two or more clones. Notably, one recurrent mutation, S698F in FptA, was observed in four clones. These findings suggest that convergent microevolution and adaption of P. aeruginosa occur in long-term ICU patients. IMPORTANCE Pseudomonas aeruginosa is a predominant opportunistic pathogen that causes nosocomial infections. Inappropriate empirical therapy can lead to prolonged hospital stays and increased mortality. In our study of sequential P. aeruginosa isolates from inpatients, high intrahost diversity was observed, including switching of clones and the emergence of a hypermutator phenotype. Recurrently mutated genes also suggested that convergent microevolution and adaption of P. aeruginosa occur in inpatients, and genomic diversity is associated with differences in multiple-drug-resistance profiles. Taken together, our findings highlight the importance of longitudinal surveillance of nosocomial P. aeruginosa clones.

Rapid and accurate etiologic diagnosis accelerates targeted antimicrobial therapy. Metagenomic analysis has played a critical role in pathogen identification. In this study, we leveraged the advantages of both the MinION and BGISEQ-500 platforms to make a bacteriologic diagnosis from a culture-negative lung tissue sample from an immunocompromised patient with severe pneumonia. Real-time nanopore sequencing rapidly identified Klebsiella pneumoniae by an 823 bp specific sequence within 1 min. Genomic analysis further identified blaSHV-12, blaKPC-2, blaTEM-1, blaCTX-M-65, and other resistance genes. The same sample was further sequenced on the BGISEQ-500 platform, which presented consistent results regarding the most top dominant pathogens and provided additional information of resistance genes. Revised antibiotic treatment was followed by the patient's clinical recovery. Though sample preparation and the interpretation of final results still need to be improved further, metagenomic sequencing contributes to the accurate diagnosis of culture-negative infections and facilitates the rational antibiotic therapy.

Background Enterobacter cloacae is an opportunistic pathogen which is responsible for serious nosocomial infections. A gene which plays an important role in resistance to carbapenems is the New Delhi metallo-β-lactamase 1 (NDM-1). Currently, the spread of NDM-1-producing E. cloacae strains is a serious public threat. Methods A multidrug-resistant E. cloacae ssp. dissolvens strain CBG15936 was recovered in 2017 in Guangzhou, China. PCR, S1-pulsed-field gel electrophoresis, and Southern blotting were performed to locate the blaNDM–1 gene. Susceptibility testing and conjugation experiments were also performed. Illumina HiSeq and Nanopore sequencers were used to perform whole-genome sequencing. Results Strain CBG15936 belongs to ST932 and is resistant to carbapenems. The blaNDM–1 gene was found on a ∼62-kb plasmid, which has a conjugation frequency of 1.68 × 10–3 events per donor cell. Genome sequencing and analysis revealed that the NDM-1-carrying IncN1 plasmid contained a new transposon Tn6696, which consists of an intact qnrS1-carrying Tn6292 element, an inverted 8.3-kb Tn3000 remnant, ISkpn19, ΔtnpA, and IS26. Conclusion A new transposon, Tn6696, has been detected on a blaNDM–1-carrying plasmid recovered from multidrug-resistant E. cloacae ssp. dissolvens CBG15936 from China. This finding provides a new perspective regarding the potential for blaNDM–1 to undergo horizontal transfer among drug-resistant bacteria.