Routine culture and PCR-based molecular testing in the clinical microbiology laboratory are unable to recognize pathogens at the strain level and to detect strain-specific genetic determinants involved in virulence and resistance. To address this issue, we explored the strain-level profiling of K. pneumoniae prevalent in China based on metagenome-sequenced patient materials.

Background: The diagnostic assay leveraging multiple reverse transcription loop-mediated isothermal amplification (RT-LAMP) could meet the requirements for rapid nucleic acid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Methods: The devised assay merged the lateral flow assay with the RT-LAMP technology and designed specific primers for the simultaneous detection of the target and human-derived internal reference genes within a single reaction. An inquiry into the assay's limit of detection (LOD), sensitivity, and specificity was carried out. The effectiveness of this assay was validated using 498 clinical specimens.Results: This LOD of the assay was determined to be 500 copies/mL, and there was no observed cross-reaction with other respiratory pathogens. The detection results derived from clinical specimens showed substantial concordance with those from real-time reverse transcription-polymerase chain reaction (RT-qPCR) (Cohen's kappa, 0.876; 95% CI: 0.833-0.919;p<0.005). The diagnostic sensitivity and specificity were 87.1% and 100%, respectively.Conclusion: The RT-LAMP assay, paired with a straightforward and disposable lateral immunochromatographic strip, achieves visual detection of dual targets for SARS-CoV-2 immediatly. Moreover, the entire procedure abstains from nucleic acids extraction. The samples are lysed at room temperature and subsequently proceed directly to the RT-LAMP reaction, which can be executed within 30 minutes at a constant temperature of 60-65°C. Then, the RT-LAMP amplification products are visualized using colloidal gold test strips.Trial registration: This study was registered at the Chinese Clinical Trial Registry (Registration number: ChiCTR2200060495, Date of registration 2022-06-03).

Introduction: Klebsiella michiganensis, a significant member of the Klebsiella oxytoca complex, has emerged as a potential pathogen in clinical settings. Despite extensive research on the Klebsiella pneumoniae complex, the pathogenicity and drug resistance of the K. oxytoca complex remain understudied, particularly regarding the reconstruction of whole genomes from metagenomic next-generation sequencing (mNGS) data.Methods: In this study, bronchoalveolar lavage fluid (BALF) from a 55-year-old woman with a suspected right lung infection in Anhui Province, China, was analyzed using mNGS.Results: Three distinct assembly strategies were employed to reconstruct the genome of K. michiganensis, leading to the identification of a novel ST452 strain, KMLRT2206. Comprehensive genomic analysis of this strain and 206 clinical isolates (genomes downloaded from public databases) revealed the population structure, distribution of drug resistance genes, and virulence factors of K. michiganensis. The results demonstrated significant genetic diversity, with the species divided into three major clades, each exhibiting distinct patterns of drug resistance and virulence genes. Notably, 38.6% of the strains harbored the bla_OXY–1–1 gene, highlighting a potential threat of drug resistance. While virulence gene distribution was not correlated with sequence type (ST), significant differences were observed among clades.Conclusion: This study underscores the value of mNGS combined with optimized assembly strategies for accurate species identification within the K. oxytoca complex, providing critical insights for clinical pathogen detection and epidemiological surveillance.

Chlamydia psittaci, a strictly intracellular bacterium, is an underestimated etiologic agent leading to infections in a broad range of animals and mild illness or pneumonia in humans. In this study, the metagenomes of bronchoalveolar lavage fluids from the patients with pneumonia were sequenced and highly abundant C. psittaci was found. The target-enriched metagenomic reads were recruited to reconstruct draft genomes with more than 99% completeness. Two C. psittaci strains from novel sequence types were detected and these were closely related to the animal-borne isolates derived from the lineages of ST43 and ST28, indicating the zoonotic transmissions of C. psittaci would benefit its prevalence worldwide. Comparative genomic analysis combined with public isolate genomes revealed that the pan-genome of C. psittaci possessed a more stable gene repertoire than those of other extracellular bacteria, with ~90% of the genes per genome being conserved core genes. Furthermore, the evidence for significantly positive selection was identified in 20 virulence-associated gene products, particularly bacterial membrane-embedded proteins and type three secretion machines, which may play important roles in the pathogen-host interactions. This survey uncovered novel strains of C. psittaci causing pneumonia and the evolutionary analysis characterized prominent gene candidates involved in bacterial adaptation to immune pressures. The metagenomic approach is of significance to the surveillance of difficult-to-culture intracellular pathogens and the research into molecular epidemiology and evolutionary biology of C. psittaci.

Acinetobacter nosocomialis is a prevalent opportunistic pathogen that causes hospital-acquired infections. The increasing threats from A. nosocomialis infections have led to attention from the scientific and medical communities. Metagenomic next-generation sequencing (mNGS) was performed for an exudate specimen collected from an ICU patient with wound infection, followed by sepsis, in Tongji Hospital. Three assembly strategies were employed to recover the genome of A. nosocomialis in the metagenomic sample. Together with publicly available genomes of A. nosocomialis, the features of population genetics and molecular epidemiology were deeply analyzed. A draft genome was reconstructed for the metagenomic strain WHM01, derived from the ST410 A. nosocomialis dominating the microbial community, thereby prompting its highly pathogenic risk, which is associated with infection and persistence. The structure of the bacterial pangenome was characterized, including the 1862 core and 11,815 accessory genes present in the 157 strains. The genetic diversity of the genes coding for the 128 virulence factors assigned to 14 functional categories was uncovered in this nosocomial pathogen, such as the lipooligosaccharide, capsule, type IV pilus, and outer membrane proteins. Our work revealed genomic properties of ST410 A. nosocomialis, which is prevalent in China, and further highlighted that metagenomic surveillance may be a prospective application for evaluating the pathogenic characteristics of the nosocomial opportunistic pathogens.

Warfarin is an anticoagulant commonly used as an oral drug in preventing and treating thromboembolic diseases. The international normalized ratio (INR) is a clinical monitoring anticoagulation intensity index that adjusts the dose based on important references. In particular, INR value must be strictly monitored when warfarin is used for anticoagulation therapy in infected patients. Herein, we report a 54-year-old female patient diagnosed with pulmonary embolism and venous thrombosis of the lower limbs. After the warfarin administration, the INR was always substandard. The patient did not take other warfarin-interacting drugs or foods during the hospital stay. Metagenome next-generation sequencing suggested a bloodstream infection caused by Escherichia coli, which was further confirmed by blood culture. After meropenem administration for anti-infective treatment, the INR value rose rapidly to a standard level. Considering the lack of relevant reports, this case is the first report of potential interaction between E. coli and warfarin. Further, in patients with thromboembolic diseases complicated by infection, antibiotics should be chosen reasonably with close monitoring of the INR to avoid the interaction of warfarin and antibiotics and to ensure the effectiveness and safety of warfarin treatment.