Annals of Clinical Microbiology and Antimicrobials最新文献

Background: Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free Mycobacterium tuberculosis (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.

Methods: Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).

Results: Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, P < 0.001) and Xpert (42.9%, P < 0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, P < 0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both P < 0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.

Conclusions: The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.

Background: Polymyxin heteroresistance poses a growing challenge in antimicrobial resistance management, yet its epidemiological features and molecular basis across different bacterial species and environments remain poorly understood. This study aimed to systematically evaluate the prevalence and genomic mechanisms of heteroresistance in Escherichia coli and Klebsiella pneumoniae.

Methods: A total of 416 isolates (272 E. coli and 144 K. pneumoniae) were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polymyxin heteroresistance was characterized using population analysis profiling (PAP) with stability validation. All isolates underwent antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS) for genomic analysis. An in vitro induction model was used to track resistance evolution.

Results: Heteroresistance prevalence was significantly higher in animal-derived than human-derived E. coli (17.83% vs. 2.10%, p < 0.01). Animal-derived resistant subpopulations carried distinct mutations in the two-components systems (TCSs) phoPQ-pmrAB/D. Structural modeling indicated that a PmrB T156M substitution disrupts kinase domain integrity, potentially triggering heteroresistance. Community-associated K. pneumoniae showed lower heteroresistance rates than clinical strains (34.03% vs. 76.62%, p < 0.01), with divergent insertion sequence (IS) distributions in the mgrB gene (IS903B/ISKpn74 vs. ISKpn26/ISKpn14). When challenged with polymyxin pressure, K. pneumoniae exhibited a significantly faster progression to polymyxin resistance than E. coli.

Conclusions: This study emphasizes the persistent influence of historical agricultural polymyxin use on the development of heteroresistance among E. coli and K. pneumoniae with diverse sources. The complexity of heteroresistance requires more cautious antibiotic selection and tailored therapeutic strategies in clinical practice to combat antimicrobial resistance.

Background: Paediatric tuberculosis (TB) diagnosis remains a challenge due to the paucibacillary nature of the disease, resulting in 51.00% of TB cases remaining undiagnosed, rising to 58% in children under five years of age.

Methods: This study evaluates the effectiveness of different microbiological methods (Xpert^® MTB/RIF Ultra, Anyplex™ MTB/NTM, culture and microscopy) for TB detection in gastric aspirate (GA) samples collected from 483 paediatric patients in Slovakia and the Czech Republic. The sensitivity of each diagnostic method was further analyzed according to patient age.

Results: The highest sensitivity was observed with the Anyplex MTB/NTM assay (38.94%). Interestingly, Xpert MTB/RIF Ultra exhibited lower sensitivity than culture (16.67% versus 21.27%). Considering all methods together, the positivity was not significantly associated with age. The overall positivity rate of all diagnostic methods combined was higher in children under five years (46.7%) and adolescents aged 16-18 years (54.2%) compared with those aged 6-15 years (31.2%). Similarly, the sensitivity of individual diagnostic methods (except Xpert MTB/RIF Ultra) followed the same trend.

Conclusion: These data indicate that gastric aspirate microbiology provides only moderate confirmation of paediatric TB in routine practice, and that nucleic acid amplification-based technologies (NAAT) should be used as part of a complementary diagnostic strategy alongside culture and smear microscopy, with culture remaining essential for drug susceptibility testing. Because NAAT platforms were applied in different centre-specific cohorts, the findings should be interpreted as real-world yields rather than a head-to-head comparison, and future work should prioritise standardised sampling and evaluation of non-invasive alternatives (e.g., stool) across age groups.

Infections caused by Carbapenem-Resistant Klebsiella pneumoniae (CRKP) have become a serious threat to global public health. We reported fatal infections associated with Klebsiella pneumoniae (KP) and revealed the evolution of in-host drug resistance that occurred during tigecycline, eravacycline and Polymyxin B treatments. In this study, six strains of KP were isolated from one patient with liver cirrhosis and chronic liver failure. Among them, one strain is Carbapenem-Sensitive Klebsiella pneumoniae (CSKP) and five strains are CRKP cloned from ST11-KL64. Polymyxin B drug sensitivity tests were conducted on strains d5, d13, d15, d17 and d21. It was found that d5 and d15 were sensitive to Polymyxin B, while strains d13, d17 and d21 were resistant to Polymyxin B. The results of drug sensitivity were consistent with the expression level of the colistin resistance gene pmrB and also consistent with the SNP difference results of pmrB in these five strains. These results further prove that the mutation site of the Polymyxin B pmrB resistance gene in this study is the Thr mutation at position 469 to Pro, which is a new mutation mechanism of the Polymyxin B resistance gene. The drug sensitivity results of eravacycline and tigecycline were consistent with their SNP difference results. It was found that position 523 on the ABC efflux pump system mutated from Ala to Thr, suggesting that the evolution of drug resistance of tigecycline and eravacycline may be related to the ABC efflux pump. The results indicated that Klebsiella pneumoniae witnessed the evolution of drug resistance in the host during the treatment with Polymyxin B, eravacycline and tigecycline, posing a potential threat to clinical anti-infective treatment.

Tuberculosis is a respiratory infectious disease that persists worldwide, largely due to the robustness and ease of dissemination of its causative agent, Mycobacterium tuberculosis. To mitigate the spread of infection, various measures have been developed to inactivate the bacteria in its aerosol form. Advanced photohydrolysis technology (APHT) was previously reported to inactivate both respiratory viral and bacterial pathogens; however, its efficacy against M. tuberculosis has yet to be evaluated. In this study, we assessed the ability of APHT to inactivate aerosolized M. tuberculosis. The bacteria were aerosolized into a custom chamber containing an APHT device, which was operated for 1 and 10 min after aerosolization. A control device lacking the APHT component was used for comparison. The APHT device achieved a 1.3-log₁₀ reduction (approximately 95%) in bacterial load after 1 min and a 2.26-log₁₀ reduction (over 99%) after 10 min. This study demonstrates the ability of APHT to inactivate aerosolized M. tuberculosis and supports its application as a possible effective infection control intervention.

Background: Respiratory viral infections remain a global health concern, particularly among children, the elderly, and immunocompromised individuals. Although real-time polymerase chain reaction (RT-PCR) is the diagnostic gold standard, its limitations in strain-level typing and mutation tracking highlight the need for complementary approaches such as next-generation sequencing (NGS).

Methods: We compared a hybridization-based NGS respiratory virus panel (RVP) in comparison with RT-PCR using 81 nasopharyngeal swab (NPS) specimens. The performance metrics included concordance rates, cycle threshold (Ct)-based stratification, co-infection detection, and strain-level classification.

Results: Among the 81 NPS specimens, RT-PCR identified respiratory viruses in 56 cases, including eight co-infections. Excluding co-infections, RVP showed 74.5% positive percent agreement, 92.3% negative percent agreement, and 80.8% overall accuracy. The detection and positive concordance rates declined with higher Ct values, and the sequencing depth also decreased. In co-infections, RVP failed to detect low-titer viruses. Strain-level classification was achieved in 65.5% of the positive samples, by subtyping rhinovirus A and C, respiratory syncytial virus A and B, and influenza A (H1N1 and H3N2).

Conclusions: NGS panel tests complement RT-PCR by enabling viral detection and strain typing, thereby offering added value to genomic surveillance and outbreak investigations.

Background: Proteus mirabilis has emerged as an important multidrug-resistant opportunistic pathogen, with the production of metallo-β-lactamases (MBLs) being a major contributor to its broad-spectrum resistance. Although the aztreonam-avibactam (ATM-AVI) combination represents a key therapeutic option against MBL-producing Enterobacteriaceae, the mechanisms underlying ATM-AVI resistance in P. mirabilis has not yet been reported.

Methods: A total of 176 multidrug-resistant P. mirabilis isolates were collected from a tertiary hospital in China (2017-2024). Antimicrobial susceptibility testing identified ATM-AVI-resistant isolates (MIC ≥ 8/4 µg/mL). Whole-genome sequencing, gene cloning, RT-qPCR, and copy number analyses were used to determine resistance mechanisms. Growth rate assays evaluated fitness costs, and global phylogenetic analysis elucidated evolutionary and dissemination patterns.

Results: Twelve isolates (6.8%, 12/176) were resistant to ATM-AVI, all carrying the bla_PER-4 gene. Cloning experiments confirmed that bla_PER-4 conferred significantly higher ATM-AVI resistance than bla_PER-1. Increased resistance correlated with bla_PER-4 overexpression and gene copy number amplification. Whole-genome analysis showed that bla_PER-4 was embedded in ISCR1-associated class 1 integrons located on both plasmids and chromosomes, with a strain carrying eight tandem chromosomal copies. These structures likely mediated gene amplification via rolling-circle replication and homologous recombination. Phylogenetic analysis revealed that bla_PER-4-positive isolates were mainly associated with the ST135 lineage, suggesting transmission event within hospitals. Global data demonstrated that bla_PER-4-carrying P. mirabilis strains were predominantly found in China (80%, 12/15), while bla_PER-1 strains were more common in the United States.

Conclusions: The bla_PER-4-carrying P. mirabilis, particularly the ST135 clone, represents a high-risk lineage associated with high-level ATM-AVI resistance mediated by gene overexpression and copy number amplification. This finding highlights a novel mechanism of ATM-AVI resistance and underscores the need for continuous genomic surveillance and rational antimicrobial stewardship to prevent its further dissemination.

Background: In this study, we were aimed to investigate the impact of co-cultures of different bacterial species on bacterial antibiotic resistance and virulence.

Methods: The effect of co-cultures of Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) on antibiotic resistance, virulence and biofilm formation in P. aeruginosa was examined in vitro in 14 mixtures. These mixtures were categorized into three groups: Standard category (including standard strains), naturally co-isolated category (co-isolated from the same patient) and random co-culture category (bacterial species from different patients). Additionally, the impact of the standard category on pathogenicity was assessed in vivo using mouse model. Intergroup comparisons were conducted using multiple t-test and comparisons between treated and untreated control isolates grown under the same conditions were made. Survival experiments were analyzed using Mantel-Cox log-rank test.

Results: P. aeruginosa survival significantly increased in most of the co-culture mixtures when treated with meropenem (92.9%), ceftazidime (85.7%), cefepime (78.6%), gentamicin (78.6%) and ciprofloxacin (71.4%). Similarly, virulence factor production significantly increased in P. aeruginosa in most of the investigated co-cultures as follows: pyocyanin (71.4%), elastase (71.4%), protease (78.6%), hemolysin (71.4%), lecithinase (78.6%), gelatinase (63.6%) and biofilm (71.4%). At the molecular level, the relative expression of the tested virulence-encoding genes; pelA, lasB and lasA were significantly increased in at least 92.9% of the co-culture mixtures, especially in random ones, compared to their mono-culture, but with varying up-regulation degree (ranging from 1.5 to 96-fold increase).

Conclusion: Finally, in vitro investigations for antibiotic resistance and virulence production clearly demonstrated a synergistic interaction between P. aeruginosa and S. aureus in the co-existence mixture. Compared to P. aeruginosa mono-cultures, the co-cultured strains exhibited enhanced resistance profiles and increased expression of key virulence factors, indicating that the simultaneous presence of both species promotes mutual adaptation and potentiation of pathogenic traits. Additionally, in vivo experiments confirmed that the co-infection with S. aureus significantly enhanced the pathogenicity of P. aeruginosa, as indicated by increased mortality rates and higher bacterial counts in lung tissues. Altogether, our results shed light on the impact of the co-existence of microbial species on bacterial virulence and antibiotic resistance.