Highly effective isolation of <i>Stenotrophomonas</i> from pharyngeal swabs of infected inpatients and issues in species identification

<i>Stenotrophomonas</i> is a significant opportunistic pathogen associated with high attributable mortality. Notably high isolation rates and species diversity have been observed among tuberculosis patients in Beijing, China. To determine its infection/colonization rate and characteristics in non-TB patients, we conducted a study utilizing a sensitive pre-enrichment PCR assay on pharyngeal swabs from inpatients in the Department of Infectious Diseases. Three <i>Stenotrophomonas</i> strains were isolated from 143 samples (2.10%), and positive cases shared common characteristics-advanced age, invasive procedures, repeated hospitalizations, and broad-spectrum antimicrobial exposure-which are established risk factors for <i>Stenotrophomonas maltophilia</i> infection. Both the isolation rate and common characteristics differed from those of TB patients. Notably, three isolates were identified via average nucleotide identity (ANI) as <i>S. geniculate</i> and <i>S. muris</i>, but both species were misidentified as <i>S. maltophilia</i> by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, despite achieving reliable identification scores (≥2.0). This misidentification likely explains the exclusive focus on <i>S. maltophilia</i> and the underestimation of other species absent from MALDI-TOF databases. Antibiotic susceptibility testing showed general susceptibility to quinolones, tigecycline, chloramphenicol, and trimethoprim-sulfamethoxazole and partial susceptibility to ceftazidime and ceftazidime/avibactam. Notably, a strain susceptible to ceftazidime and moxifloxacin was isolated from a patient treated with these agents. In conclusion, this study underscores the necessity of developing alternative rapid molecular methods with ANI-equivalent resolution for <i>Stenotrophomonas</i>, alongside expanding mass spectral databases and reevaluating the clinical significance and resistance profiles of the <i>Stenotrophomonas</i> species.IMPORTANCECompounding its inherent pathogenicity, <i>Stenotrophomonas</i> adversely impacts co-infecting pathogens and is associated with high attributable mortality and multidrug resistance. This study demonstrates that through enrichment and PCR of throat swab samples, this elusive pathogen-frequently undetected in routine clinical testing-can be identified with high sensitivity, providing crucial diagnostic alerts for clinicians, particularly in critically ill and etiology-unknown patients. Our analysis reveals that the risk factors for <i>Stenotrophomonas</i> positivity differ between general infectious disease inpatients and tuberculosis patients. Furthermore, our findings highlight a critical issue of species misidentification by routine microbiological methods, which has obscured the true clinical significance and resistance profiles of <i>Stenotrophomonas</i> species.